Sheath fluid systems and methods for particle analysis in blood samples

ABSTRACT

Aspects and embodiments of the instant disclosure provide a particle and/or intracellular organelle alignment agent for a particle analyzer used to analyze particles contained in a sample. An exemplary particle and/or intracellular organelle alignment agent includes an aqueous solution, a viscosity modifier, and/or a buffer.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/064,129, filed on Mar. 8, 2016, entitled “SHEATH FLUID SYSTEMS ANDMETHODS FOR PARTICLE ANALYSIS IN BLOOD SAMPLES,” which is a divisionalof U.S. patent application Ser. No. 14/215,834, filed on Mar. 17, 2014,entitled SHEATH FLUID SYSTEMS AND METHODS FOR PARTICLE ANALYSIS IN BLOODSAMPLES,” which is a non-provisional of, and claims the benefit ofpriority to, U.S. Provisional Patent Application No. 61/799,152 filedMar. 15, 2013, the content of each of which is incorporated herein byreference. This application is also related to U.S. patent applicationSer. Nos. 14/216,811, 14/216,533 (issued as U.S. Pat. No. 9,322,752),Ser. Nos. 14/217,034, and 14/216,339 and PCT International PatentApplication Nos. PCT/US14/30928, PCT/US14/30902, and PCT/US14/30851, allfiled Mar. 17, 2014. The content of each of these filings isincorporated herein by reference.

BACKGROUND OF THE INVENTION

This disclosure relates to the field of apparatus, systems,compositions, and methods for analysis of particles, including imagingof particles in fluid samples, using wholly or partly automated devicesto discriminate and quantify particles such as blood cells in thesample. The present disclosure also relates to a particle and/orintracellular organelle alignment liquid (PIOAL) useful for analyzingparticles in a sample from a subject, methods for producing the liquid,and methods for using the liquid to detect and analyze particles.Compositions, systems, devices and methods useful for conductingimage-based biological fluid sample analysis are also disclosed. Thecompositions, systems, devices, and methods of the present disclosureare also useful for detecting, counting and characterizing particles inbiological fluids such as red blood cells, reticulocytes, nucleated redblood cells, platelets, and for image and morphologically-based whiteblood cell differential counting, categorization, subcategorization,characterization and/or analysis.

Blood cell analysis is one of the most commonly performed medical testsfor providing an overview of a patient's health status. A blood samplecan be drawn from a patient's body and stored in a test tube containingan anticoagulant to prevent clotting. A whole blood sample normallycomprises three major classes of blood cells including red blood cells(erythrocytes), white blood cells (leukocytes) and platelets(thrombocytes). Each class can be further divided into subclasses ofmembers. For example, five major types or subclasses of white bloodcells (WBCs) have different shapes and functions. White blood cells mayinclude neutrophils, lymphocytes, monocytes, eosinophils, and basophils.There are also subclasses of the red blood cell types. The appearancesof particles in a sample may differ according to pathologicalconditions, cell maturity and other causes. Red blood cell subclassesmay include reticulocytes and nucleated red blood cells.

A blood cell count estimating the concentration of RBCs, WBCs orplatelets can be done manually or using an automatic analyzer. Whenblood cell counts are done manually, a drop of blood is applied to amicroscope slide as a thin smear. Traditionally, manual examination of adried, stained smear of blood on a microscope slide has been used todetermine the number or relative amounts of the five types of whiteblood cells. Histological dyes and stains have been used to stain cellsor cellular structures. For example, Wright's stain is a histologicstain that has been used to stain blood smears for examination under alight microscope. A Complete Blood Count (CBC) can be obtained using anautomated analyzer, one type of which counts the number of differentparticles or cells in a blood sample based on impedance or dynamic lightscattering as the particles or cells pass through a sensing area along asmall tube. The automated CBC can employ instruments or methods todifferentiate between different types of cells that include RBCs, WBCsand platelets (PLTs), which can be counted separately. For example, acounting technique requiring a minimum particle size or volume might beused to count only large cells. Certain cells such as abnormal cells inthe blood may not be counted or identified correctly. Small cells thatadhere to one another may be erroneously counted as a large cell. Whenerroneous counts are suspected, manual review of the instrument'sresults may be required to verify and identify cells.

Automated blood cell counting techniques can involve flow cytometry.Flow cytometry involves providing a narrow flow path, and sensing andcounting the passage of individual blood cells. Flow cytometry methodshave been used to detect particles suspended in a fluid, such as cellsin a blood sample, and to analyze the particles as to particle type,dimension, and volume distribution so as to infer the concentration ofthe respective particle type or particle volume in the blood sample.Examples of suitable methods for analyzing particles suspended in afluid include sedimentation, microscopic characterization, countingbased on impedance, and dynamic light scattering. These tools aresubject to testing errors. On the other hand, accurate characterizationof types and concentration of particles may be critical in applicationssuch as medical diagnosis.

In counting techniques based on imaging, pixel data images of a preparedsample that may be passing through a viewing area are captured using amicroscopy objective lens coupled to a digital camera. The pixel imagedata can be analyzed using data processing techniques, and alsodisplayed on a monitor.

Aspects of automated diagnosis systems with flowcells are disclosed inU.S. Pat. No. 6,825,926 to Turner et al. and in U.S. Pat. Nos.6,184,978; 6,424,415; and 6,590,646, all to Kasdan et al., which arehereby incorporated by reference as if set forth fully herein.

Automated systems using dynamic light scattering or impedance have beenused to obtain a complete blood count (CBC): total white blood cellcount (WBC), total cellular volume of red blood cells (RBCdistribution), hemoglobin HGB (the amount of hemoglobin in the blood);mean cell volume (MCV) (mean volume of the red cells); MPV (mean PLTvolume); hematocrit (HCT); MCH (HGB/RBC) (the average amount ofhemoglobin per red blood cell); and MCHC (HGB/HCT) (the averageconcentration of hemoglobin in the cells). Automated or partiallyautomated processes have been used to facilitate white blood cell fivepart differential counting and blood sample analyses.

Although such currently known particle analysis systems and methods,along with related medical diagnostic techniques, can provide realbenefits to doctors, clinicians, and patients, still furtherimprovements are desirable. For example, there is a continuing need forimproved methods and compositions useful for particle and/orintracellular organelle alignment when performing image-based sampleanalysis using automated systems. Embodiments of the present inventionprovide solutions for at least some of these outstanding needs.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the present invention relate to apparatus, systems,compositions, and methods for analyzing a prepared sample containingparticles. In some aspects the system comprises an analyzer which may bea visual analyzer. In some aspects, the apparatus contains a visualanalyzer and a processor. In one aspect, this disclosure relates to anautomated particle imaging system in which a liquid sample containingparticles of interest is caused to flow through a flowcell having aviewport through which a high optical resolution imaging device capturesan image. In some aspects the high optical resolution imaging devicecomprises a camera such as a digital camera. In one aspect the highoptical resolution imaging device comprises an objective lens.

The flowcell is coupled to a source of sample fluid, such as a preparedsample, and to a source of particle and/or intracellular organellealignment liquid (PIOAL). The system permits capture of focused imagesof particles in a sample in flow. In some embodiments the images can beused in automated, high throughput processes for categorizing andsubcategorizing particles. An exemplary visual analyzer may include aprocessor to facilitate automated analysis of the images. In some cases,the visual analyzer can be used in methods of this disclosure to provideautomated image-based WBC differential counting or other blood sampleparticle analysis protocols. In some cases, the methods of thisdisclosure relate to automated identification of morphologicalabnormalities for determining, diagnosing, prognosing, predicting,and/or supporting a diagnosis of whether a subject is healthy or has adisease, condition, abnormality and/or infection and for monitoringwhether a subject is responsive or non-responsive to treatment.

Embodiments of the present invention provide systems, methods, andsheath fluid compositions useful for particle and/or intracellularorganelle alignment in cells treated with particle contrast agentcompositions. Such techniques overcome certain difficulties associatedwith conventional sheath fluids used in flow cytometry that may sufferfrom the disadvantages of maintaining cell morphology and/or notproviding for the capture of optimized images which permit determinationof one or more blood components.

In certain embodiments, a viscosity difference and/or speed differencebetween a ribbon-shaped sample stream and a sheath fluid and/or athickness of the ribbon-shaped sample stream, for example in combinationwith a geometric focusing effect provided by a narrowing flowpathtransition zone, can introduce shear forces to act on the particleswhile in flow thereby causing the particles to align or remain inalignment throughout an imaging process in a visual analyzer. In someembodiments the sample will be contrast enhanced. In some embodimentsthe sheath fluid may comprise up to 100% of a viscosity agent. Inanother embodiment, the sheath fluid has up to 60% v/v of a viscosityagent. Depending on the types of viscosity agent used, in someembodiments the sheath fluid may comprise a viscosity agent that iscommercially available in dry form at a concentration of about 5 to 7%,or more specifically at 6.5% (w/v).

In other embodiments, this disclosure relates to a sheath fluid that canbe used in image based analysis of particles in samples such as cellsand other particle features in other biological fluids such ascerebrospinal fluid and effusions associated with particular conditions.Cell category and/or subcategory counts as described for use in bloodsamples in this disclosure as nonlimiting examples of the sort ofsamples that may be analyzed. In some embodiments, cells present insamples may also include bacterial or fungal cells as well as whiteblood cells, red blood cells or platelets. In some embodiments, particlesuspensions obtained from tissues or aspirates may be analyzed.

In some embodiments a stream of sample fluid can be injected through acannula with a flattened opening to establish a flowpath with aconsiderable width. The sheath fluid can be introduced into the flowcelland carries the sample fluid along through the imaging area, then towarda discharge. A sheath fluid has a different viscosity, e.g., higher,than the sample fluid, and, optionally, a different flow rate at thepoint of injection to the ribbon-shaped sample stream results in thesample fluid flattening into a thin ribbon shape. The thin ribbon ofsample fluid is carried along with the sheath fluid, through a narrowingflowpath transition zone, to pass in front of a viewing port where ahigh optical resolution imaging device and a light source are arrangedto view the ribbon-shaped sample stream.

In one embodiment, the viscosity of the sheath fluid can be higher thanthe viscosity of the sample. The viscosity of the sheath fluid, theviscosity of the sample material, the flow rate of the sheath fluid andthe flow rate of the sample material are coordinated, for example incombination with a ribbon compression effect provided by a narrowingtransition zone, to provide the flow in a ribbon-shaped sample streamwith predetermined dimensional characteristics, such as an advantageousribbon-shaped sample stream thickness. Maintaining an advantageousribbon-shaped sample stream thickness provides, as an example, a highpercentage of in-focus cells or in-focus cellular components.

Embodiments of the instant disclosure are based at least in part on thediscovery that the addition of a suitable amount of a viscosity agent inthe sheath fluid significantly improves particle/cell alignment in aflowcell, for example in a flowcell having a narrowing transition zone,and increases in-focus intracellular contents of cells, resulting inhigher quality images of cells in flow compared to use of a nonviscosity-modified conventional sheath fluid used in flow cytometry. Theaddition of the viscosity agent increases the shear forces on elongateor nonspherical particles or cells like red blood cells (RBCs) whichthen aligns the cells in a plane substantially parallel to the flowdirection, which results in image optimization. For cells like whiteblood cells (WBCs), this also results in positioning, repositioning,and/or better-positioning of intracellular structures, organelles orlobes substantially parallel to the direction of flow. For example, thewhite blood cells can be compressible or deformable in response to theshear forces conferred by the viscosity agent or differential, thusleading to particle elongation or compression and alignment under shear.

Alignment of particles that are smaller in diameter than the flow streammay be obtained by increasing the viscosity of the sheath fluid. Thisresults in improved alignment of those particles in a planesubstantially parallel to the direction of the flow.

The ribbon-shaped sample stream thickness can be affected by therelative viscosities and flow rates of the sample fluid and the sheathfluid, for example in combination with the geometry of the narrowingtransition zone of the flowcell. The feed source of the sample and/orthe feed source of the sheath fluid, for example comprising precisiondisplacement pumps, can be configured to provide the sample and/or thesheath fluid at stable flow rates for optimizing the dimensions of theribbon-shaped sample stream, namely as a thin ribbon at least as wide asthe field of view of the imaging device.

An exemplary sheath fluid embodiment is used in a flowcell for particleanalysis. A sample is enveloped in the stream of the sheath fluid andpassed through the flowcell of the analyzer device. Then informationfrom the sample when passing through the detection area is collected,enabling an analyzer to analyze particles/cells contained in the sample.The use of the sheath fluid on such an analyzer allows accuratecategorization and subcategorization and counting of cells and/orparticles contained in samples.

As used herein, sheath fluid is useful in obtaining information relatingto following cells and/or particles related thereto: including forexample; neutrophil, lymphocyte, monocyte, eosinophil, basophil,platelet, reticulocyte, nucleated RBC, blast, promyelocyte, myelocyte,and/or a metamyelocyte.

The present disclosure provides novel compositions and methods of usethereof for conducting particle analysis. In particular, the presentdisclosure relates to a particle and/or intracellular organellealignment liquid (PIOAL) used in a analyzer for analyzing particles in asample. The terms sheath fluid and PIOAL can be used interchangeablythroughout this disclosure. The present disclosure further providesmethods for producing the PIOAL and methods for using the PIOAL toanalyze particles. The PIOAL of this invention is useful, as an example,in methods for automated categorization and subcategorization ofparticles in a sample.

In one aspect, embodiments of the present invention encompass methodsfor imaging a plurality of particles using a particle analysis system.The system can be configured for combined viscosity and geometrichydrofocusing. The particles can be included in a blood fluid samplehaving a sample fluid viscosity. Exemplary methods can include flowing asheath fluid along a flowpath of a flowcell, and the sheath fluid canhave a sheath fluid viscosity that differs from the sample fluidviscosity by a viscosity difference in a predetermined viscositydifference range. Methods can also include injecting the blood fluidsample into the flowing sheath fluid within the flowcell so as toprovide a sample fluid stream enveloped by the sheath fluid. Further,methods can include flowing the sample fluid stream and the sheath fluidthrough a reduction in flowpath size toward an imaging site, such that aviscosity hydrofocusing effect induced by an interaction between thesheath fluid and the sample fluid stream associated with the viscositydifference, in combination with a geometric hydrofocusing effect inducedby an interaction between the sheath fluid and the sample fluid streamassociated with the reduction in flowpath size, is effective to providea target imaging state in at least some of the plurality of particles atthe imaging site while a viscosity agent in the sheath fluid retainsviability of cells in the sample fluid stream leaving structure andcontent of the cells intact when the cells extend from the sample fluidstream into the flowing sheath fluid. What is more, methods may includeimaging the plurality of particles at the imaging site. In some cases,the sheath fluid has an index of refraction n=1.3330. In some cases, thesheath fluid has an index of refraction that is the same as the index ofrefraction of water. In some cases, the interaction between the sheathfluid and the sample fluid stream associated with the reduction inflowpath size contributes to providing the target imaging state byproducing shear forces along the interfaces of the sample and sheathfluid streams. In some cases, the target imaging state includes a targetorientation of one or more target particles in the flow relative to afocal plane of an imaging device used to acquire images at the imagingsite.

According to some embodiments, the flowpath at the imaging site definesa plane that is substantially parallel to the focal plane. In somecases, the target orientation corresponds to a target alignment relativeto the focal plane at the imaging site. In some cases, the targetalignment corresponds to a target particle alignment relative to thefocal plane at the imaging site. In some cases, the target alignmentcorresponds to a target intraparticle structure alignment relative tothe focal plane at the imaging site. In some cases, the targetorientation corresponds to a target position relative to the focal planeat the imaging site. In some cases, the target position corresponds to atarget particle position relative to a focal plane at the imaging site.In some cases, the target position corresponds to a target intraparticlestructure position relative to a focal plane at the imaging site. Insome cases, the target position is within the focal plane. In somecases, the target position is at a distance from the focal plane, thedistance corresponding to a positional tolerance. In some cases, thetarget orientation corresponds to a target alignment relative to thefocal plane and a target position relative to the focal plane. In somecases, the target imaging state corresponds to a target orientation ofone or more target intraparticle structures in the flow relative to afocal plane of an imaging device used to acquire images at the imagingsite. In some cases, the flowpath at the imaging site defines a planethat is substantially parallel to the focal plane. In some cases, thetarget orientation corresponds to a target alignment relative to thefocal plane at the imaging site. In some cases, the target alignmentcorresponds to a target particle alignment relative to the focal planeat the imaging site. In some cases, the target alignment corresponds toa target intraparticle structure alignment relative to the focal planeat the imaging site. In some cases, the target orientation correspondsto a target position relative to the focal plane at the imaging site. Insome cases, the target position corresponds to a target particleposition relative to a focal plane at the imaging site. In some cases,the target position corresponds to a target intraparticle structureposition relative to a focal plane at the imaging site. In some cases,the target position is within the focal plane. In some cases, the targetposition is at a distance from the focal plane, the distancecorresponding to a positional tolerance. In some cases, the targetorientation corresponds to a target alignment relative to the focalplane and a target position relative to the focal plane. In some cases,the target imaging state corresponds to a target deformation of one ormore target particles or of one or more target intraparticle structures.

According to some embodiments, the process of injecting the blood fluidsample is performed by directing a stream of the blood fluid samplethrough a sample injection tube with a sample fluid velocity. Theinjection tube can have a port within the flowpath. The port can definea width, a thickness, and a flow axis extending along the flowpath. Thewidth can be being greater than the thickness so that the sample streamhas opposed major surfaces transverse to the imaging path adjacent theimaging site. In some cases, the sheath fluid flowing along the flowpathof the flowcell extends along the major surfaces of the sample streamand has a sheath fluid velocity different than the sample fluidvelocity. In some cases, an interaction between the sheath fluid and thesample fluid associated with the differing velocities, in combinationwith the interaction between the sheath fluid and the sample fluidassociated with the differing viscosities, provides the target imagingstate. According to some embodiments, the plurality of particles caninclude a red blood cell, a white blood cell, and/or a platelet.According to some embodiments, the plurality of particles can include acell having an intraparticle structure. In some cases, an intraparticlestructure can be an intracellular structure, an organelle, or a lobe.

In some embodiments, the sheath fluid has a viscosity between 1 and 10centipoise (cP). In some cases, the predetermined viscosity differencehas an absolute value within a range from about 0.1 to about 10centipoise (cP). In some cases, the predetermined viscosity differencehas an absolute value within a range from about 1.0 to about 9.0centipoise (cP). In some cases, the predetermined viscosity differencehas an absolute value within a range from about 1.0 to about 5.0centipoise (cP). In some cases, predetermined viscosity difference hasan absolute value of about 3.0 centipoise (cP). In some cases, theviscosity agent of the sheath fluid includes glycerol, glycerolderivative, ethylene glycol, propylene glycol (dihydroxypropane),polyethylene glycol, polyvinylpyrrolidone (PVP), carboxymethylcellulose(CMC), water soluble polymer(s), and/or dextran. In some cases, theviscosity agent of the sheath fluid includes glycerol at a concentrationbetween about 1 to about 50% (v/v). In some cases, the viscosity agentof the sheath fluid includes glycerol and polyvinylpyrrolidone (PVP). Insome cases, the viscosity agent of the sheath fluid includes glycerol ata concentration of 5% (v/v) and glycerol and polyvinylpyrrolidone (PVP)at a concentration of 1% (w/v). In some cases, the viscosity agent ofthe sheath fluid includes glycerol present at a final concentrationbetween about 3 to about 30% (v/v) under operating conditions. In somecases, the viscosity agent of the sheath fluid includes glycerol presentat a final concentration of about 30% (v/v) under operating conditions.In some cases, the viscosity agent of the sheath fluid includes glycerolpresent at a final concentration of about 6.5% v/v under operatingconditions. In some cases, the viscosity agent of the sheath fluidincludes glycerol present at a final concentration of about 5% (v/v) andpolyvinylpyrrolidone (PVP) present at a concentration of about 1% (w/v)under operating conditions.

According to some embodiments, the blood fluid sample at the imagingsite has a linear velocity within a range from 20 to 200 mm/second. Insome cases, the blood fluid sample at the imaging site has a linearvelocity within a range from 50 to 150 mm/second. In some cases, theblood fluid sample has a sample stream thickness of up to 7 μm and asample stream width within a range from 500 to 3000 μm at the imagingsite. In some cases, the blood fluid sample has sample stream thicknesswithin a range from 2 to 4 μm and a sample stream width within a rangefrom 1000 to 2000 μm at the imaging site. In some cases, the pluralityof particles includes a set of non-spherical particles, the blood fluidsample has a direction of flow at the imaging site, and more than 75% ofthe set of non-spherical particles are aligned substantially in a planeparallel to the direction of flow such a major surface of each alignednon-spherical particle is parallel to the plane parallel to thedirection of flow. In some cases, the plurality of particles includes aset of non-spherical particles, the blood fluid sample has a directionof flow at the imaging site, and at least 90% of the set ofnon-spherical particles are aligned within 20 degrees from a planesubstantially parallel to the direction of flow. In some cases, theplurality of particles includes intraparticle structures, the bloodfluid sample has a direction of flow at the imaging site, and at least92% of the intraparticle structures are substantially parallel to thedirection of flow.

In another aspect, embodiments of the present invention encompasssystems for imaging a plurality of particles in a blood fluid samplehaving a sample fluid viscosity. The system can be configured for usewith a sheath fluid having a sheath fluid viscosity that differs fromthe sample fluid viscosity by a viscosity difference in a predeterminedviscosity difference range. Exemplary systems can include a flowcellhaving a flowpath and a sample fluid injection tube, the flowpath havinga reduction in flowpath size, a sheath fluid input in fluidcommunication with the flowpath of the flowcell so as to transmit a flowof the sheath fluid along the flowpath of the flowcell, and a bloodfluid sample input in fluid communication with the injection tube of theflowcell so as to inject a flow of the blood fluid sample into theflowing sheath fluid within the flowcell, such that as the sheath fluidand the sample fluid flow through the reduction in flowpath size andtoward an imaging site, a viscosity hydrofocusing effect induced by aninteraction between the sheath fluid and the sample fluid associatedwith the viscosity difference, in combination with a geometrichydrofocusing effect induced by an interaction between the sheath fluidand the sample fluid associated with the reduction in flowpath size,provides a target imaging state in at least some of the plurality ofparticles at the imaging site while a viscosity agent in the sheathfluid retains viability of cells in the sample fluid stream leavingstructure and content of the cells intact when the cells extend from thesample fluid stream into the flowing sheath fluid. Systems can furtherinclude an imaging device that images the plurality of particles at theimaging site.

According to some embodiments, the target imaging state corresponds to atarget orientation of one or more target particles in the flow relativeto a focal plane of an imaging device used to acquire images at theimaging site. In some cases, the plurality of particles includes amember selected from the group consisting of a red blood cell, a whiteblood cell, and a platelet. In some cases, the plurality of particlesincludes a cell having an intraparticle structure. An intracellularstructure can be an intracellular structure, an organelle, or a lobe. Insome cases, the predetermined viscosity difference has an absolute valuewithin a range from about 0.1 to about 10 centipoise (cP). In somecases, the viscosity agent of the sheath fluid includes glycerol, aglycerol derivative, ethylene glycol, propylene glycol(dihydroxypropane), polyethylene glycol, polyvinylpyrrolidone (PVP),carboxymethylcellulose (CMC), water soluble polymer(s), and/or dextran.In some cases, the viscosity agent of the sheath fluid includes glycerolat a concentration between about 1 to about 50% (v/v). In some cases,the viscosity agent of the sheath fluid includes glycerol andpolyvinylpyrrolidone (PVP). In some cases, the viscosity agent of thesheath fluid includes glycerol at a concentration of 5% (v/v) andglycerol and polyvinylpyrrolidone (PVP) at a concentration of 1% (w/v).

According to some embodiments, the plurality of particles includes a setof non-spherical particles, the blood fluid sample has a direction offlow at the imaging site, and at least 90% of the set of non-sphericalparticles are aligned within 20 degrees from a plane substantiallyparallel to the direction of flow. In some cases, the target orientationcorresponds to a target particle orientation relative to a focal planeat the imaging site. A particle may be a red blood cell, an white bloodcell, or a platelet, in some embodiments. In some cases, the targetorientation corresponds to a target intraparticle structure orientationrelative to a focal plane at the imaging site. (e.g. intraparticlestructure can be an intracellular structure, an organelle, or a lobe).In some cases, the flowpath at the imaging site defines a plane that issubstantially parallel to the focal plane. In some cases, the targetorientation corresponds to a target alignment relative to the focalplane at the imaging site. In some cases, the target alignmentcorresponds to a target particle alignment relative to a focal plane atthe imaging site. In some cases, the target alignment corresponds to atarget intraparticle structure alignment relative to a focal plane atthe imaging site. In some cases, the target orientation corresponds to atarget position relative to the focal plane at the imaging site. In somecases, the target position corresponds to a target particle positionrelative to a focal plane at the imaging site. In some cases, the targetposition corresponds to a target intraparticle structure positionrelative to a focal plane at the imaging site. In some cases, the targetposition is within the focal plane. In some cases, the target positionis at a distance from the focal plane, the distance corresponding to apositional tolerance. In some cases, the target orientation correspondsto a target alignment relative to the focal plane and a target positionrelative to the focal plane. In some cases, the target imaging statecorresponds to a target deformation at the imaging site.

According to some embodiments, a blood fluid sample source can beconfigured to provide the blood fluid sample a sample fluid velocityinto the flowing sheath fluid, such that the sheath fluid has a sheathfluid velocity that is different from the sample fluid velocity. In somecases, an interaction between the sheath fluid and the sample fluidassociated with the differing velocities, in combination with theinteraction between the sheath fluid and the sample fluid associatedwith the differing viscosities, provides the target imaging state.

According to some embodiments, the flowpath of the flowcell includes azone with a change in flowpath size, and an interaction between thesheath fluid and the sample fluid associated with the change in flowpathsize, in combination with the interaction between the sheath fluid andthe sample fluid associated with the differing viscosities, provides thetarget imaging state. In some cases, the interaction between the sheathfluid and the sample fluid associated with the change in flowpath sizecontributes to providing the target imaging state by producing a lateralfluid compression force. In some cases, the plurality of particlesincludes a red blood cell, a white blood cell, and/or a platelet. Insome cases, the plurality of particles includes a cell having anintraparticle structure, and the structure can be an intracellularstructure, an organelle, or a lobe.

According to some embodiments, the predetermined viscosity differencehas an absolute value within a range from about 0.1 to about 10centipoise (cP). In some cases, the predetermined viscosity differencehas an absolute value within a range from about 1.0 to about 9.0centipoise (cP). In some cases, the predetermined viscosity differencehas an absolute value within a range from about 1.0 to about 5.0centipoise (cP). In some cases, the predetermined viscosity differencehas an absolute value of about 3.0 centipoise (cP). In some cases, thesheath fluid includes a viscosity agent which can include glycerol, aglycerol derivative, ethylene glycol, propylene glycol(dihydroxypropane), polyethylene glycol, polyvinylpyrrolidone (PVP),carboxymethylcellulose (CMC), water soluble polymer(s), and/or dextran.In some cases, the sheath fluid comprises glycerol at a concentrationbetween about 1 to about 50% (v/v).

According to some embodiments, the blood fluid sample at the imagingsite has a linear velocity within a range from 20 to 200 mm/second. Insome cases, the blood fluid sample at the imaging site has a linearvelocity within a range from 50 to 150 mm/second. In some cases, theblood fluid sample has a sample stream thickness of up to 7 μm and asample stream width of over 500 μm at the imaging site. In some cases,the blood fluid sample has a sample stream thickness within a range from2 to 4 μm and a sample stream width within a range from 1000 to 2000 μmat the imaging site. In some cases, the plurality of particles includesa set of non-spherical particles, the blood fluid sample has a directionof flow at the imaging site, and at least 90% of the set ofnon-spherical particles are aligned and/or positioned substantially in aplane parallel to the direction of flow. In some cases, the plurality ofparticles includes a set of non-spherical particles, the blood fluidsample has a direction of flow at the imaging site, and at least 95% ofthe set of non-spherical particles are aligned within 20 degrees from aplane substantially parallel to the direction of flow. In some cases,the plurality of particles include intraparticle structures, the bloodfluid sample has a direction of flow at the imaging site, and at least92% of the intraparticle structures are substantially parallel to thedirection of flow.

In another aspect, embodiments of the present invention encompass aparticle and intracellular organelle alignment liquid (PIOAL) for use ina combined viscosity and geometric hydrofocusing analyzer. The PIOAL candirect flow of a blood sample fluid of a given viscosity that isinjected into a narrowing flowcell transition zone of the visualanalyzer so as to produce a sample fluid stream enveloped by the PIOAL.The PIOAL can include a fluid having a higher viscosity than theviscosity of the blood sample fluid. A viscosity hydrofocusing effectinduced by an interaction between the PIOAL fluid and the sample fluidassociated with the viscosity difference, in combination with ageometric hydrofocusing effect induced by an interaction between thePIOAL fluid and the sample fluid associated with the narrowing flowcelltransition zone, is effective to provide a target imaging state in atleast some of the plurality of particles at an imaging site of thevisual analyzer while a viscosity agent in the PIOAL retains viabilityof cells in the sample fluid stream leaving structure and content of thecells intact when the cells extend from the sample fluid stream into theflowing sheath fluid. In some cases, the viscosity agent of the sheathfluid includes glycerol, a glycerol derivative, ethylene glycol,propylene glycol (dihydroxypropane), polyethylene glycol,polyvinylpyrrolidone (PVP), carboxymethylcellulose (CMC), water solublepolymer(s), and/or dextran. In some cases, the viscosity agent of thesheath fluid includes glycerol at a concentration between about 1 toabout 50% (v/v). In some cases, the viscosity agent of the sheath fluidincludes polyvinylpyrrolidone (PVP). In some cases, thepolyvinylpyrrolidone (PVP) is at a concentration of 1% (w/v). In somecases, the viscosity agent of the sheath fluid further includesglycerol. In some cases, the viscosity agent of the sheath fluidincludes glycerol at a concentration of 5% (v/v) and glycerol andpolyvinylpyrrolidone (PVP) at a concentration of 1% (w/v). In somecases, the PIOAL has a viscosity of between about 1-10 centipoise (cP).

In yet another aspect, embodiments of the present invention encompass aparticle and intracellular organelle alignment liquid (PIOAL) for use ina visual analyzer configured to direct flow of a sample of a givenviscosity in a flow path. The PIOAL can include a fluid having a higherviscosity than the viscosity of the sample. The PIOAL can be effectiveto support the flow of the sample and to align particles and increasethe in-focus content of particles and intracellular organelles of cellsflowing in the flowpath, whereby the aligned particles and intracellularorganelles of cells can be imaged. In some cases, the PIOAL furtherincludes a viscosity agent. In some cases, the PIOAL further includes abuffer, a pH adjusting agent, an antimicrobial agent, an ionic strengthmodifier, a surfactant, and/or a chelating agent. In some cases, theparticle and intracellular organelle alignment liquid is isotonic. Insome cases, the particle and intracellular organelle alignment liquidincludes sodium chloride. In some cases, wherein the sodium chloride ispresent at a concentration of about 0.9%. In some cases, the pH of thePIOAL sample is between about 6.0 to about 8.0 under operatingconditions. In some cases, the pH of the PIOAL sample mixture is betweenabout 6.5 to about 7.5 under operating conditions. In some cases, thePIOAL includes a pH adjusting agent for adjusting the pH is betweenabout 6.8 to about 7.2 under operating conditions. In some cases, thePIOAL liquid has a target viscosity of between about 1-10 centipoiseunder operating conditions.

In still yet another aspect, embodiments of the present inventionencompass a stock solution of concentrated PIOAL. In some cases, theconcentrated stock solution can be diluted to achieve the targetviscosity. In some cases, the concentration of the stock solution ispresent at least about 1.1× to at least about 100× concentration of thePIOAL under operating conditions. In some cases, the viscosity agent isselected from at least one of glycerol, glycerol derivative; PVP, CMC,ethylene glycol; propylene glycol (dihydroxypropane); polyethyleneglycol; water soluble polymer and dextran. In some cases, the viscosityagent includes glycerol. In some cases, the viscosity agent includesglycerol and polyvinylpyrrolidone (PVP). In some cases, the viscosityagent includes glycerol and carboxymethylcellulose (CMC). In some cases,the viscosity agent includes glycerol and dextran sulfate. In somecases, the viscosity agent includes a glycerol derivative. In somecases, the viscosity agent includes PVP. In some cases, the viscosityagent includes propylene glycol (dihydroxypropane). In some cases, theviscosity agent includes polyethylene glycol. In some cases, theviscosity agent includes water soluble dextran. In some cases, theglycerol is present at a final concentration between about 1 to about50% (v/v) under operating conditions. In some cases, said glycerol ispresent at a final concentration between about 3 to about 30% (v/v)under operating conditions. In some cases, said glycerol is present at afinal concentration of about 30% (v/v) under operating conditions. Insome cases, said glycerol is present at a final concentration of about6.5% v/v under operating conditions. In some cases, said glycerol ispresent at a final concentration of about 5% v/v and the PVP is presentat a concentration of about 1% w/v under operating conditions. In somecases, said PVP is present at a final concentration of about 1% w/vunder operating conditions. In some cases, embodiments of the presentinvention encompass kits that include a PIOAL as disclosed herein.

In another aspect, embodiments of the present invention encompassmethods for analyzing a plurality of cells in a blood fluid samplehaving a sample fluid viscosity, the cells having opposed majorsurfaces. Exemplary methods can include flowing a sheath fluid along aflowpath of a flowcell. The sheath fluid can have a sheath fluidviscosity higher than the sample fluid viscosity. Methods can alsoinclude injecting the blood fluid sample into the flowing sheath fluidwithin the flowcell. The plurality of cells can include a first subsetwith major surfaces oriented transverse to an orientation of an imagingpath. Methods can also include imaging the particles along the imagingpath at an imaging site while the plurality of cells include a secondsubset with the major surfaces oriented transverse to the imaging path,the second subset being more numerous than the first subset. Methods canalso include directing the fluid blood sample and the sheath fluidthrough a reduction in flowpath size such that an interaction betweenthe sheath fluid and the sample fluid associated with the differingviscosities reorients at least some of the plurality of cells such thatthe second subset is more numerous than the first subset.

In another aspect, embodiments of the present invention encompasssystems for imaging a plurality of cells in a blood fluid sample havinga sample fluid viscosity. Systems can be configured for use with asheath fluid having a sheath fluid viscosity higher than the samplefluid viscosity, the cells having opposed major surfaces. Exemplarysystems can include a flowcell having a flowpath and a sample fluidinjection tube, a sheath fluid input in fluid communication with theflowpath of the flowcell so as to transmit a flow of the sheath fluidalong the flowpath of the flowcell, and a blood fluid sample input influid communication with the injection tube of the flowcell so as toinject a flow of the blood fluid sample into the flowing sheath fluidwithin the flowcell such that the plurality of the injected cellsincluding a first subset with major surfaces aligned transverse to anorientation of an imaging path. In some cases, the flowpath of theflowcell can have a zone with a change in flowpath size configured suchthat an interaction between the sheath fluid and the blood sample fluidassociated with the differing viscosities reorients at least some of theparticles. Systems can also include an imaging device that images theplurality of particles along the imaging path at an imaging site whilethe major surfaces of the second subset of the plurality of cells areoriented transverse to the imaging path.

In one aspect, this invention relates to a method for imaging a particlecomprising: treating particles in a sample using the particle contrastagent compositions of this disclosure; illuminating the stained particlewith light in a visual analyzer comprising a flowcell and autofocusapparatus; obtaining a digitized image of the particle enveloped in aparticle and/or intracellular organelle alignment liquid (PIOAL); and;analyzing a particle in the sample based on the image information. Insome embodiments, the particle is selected from at least one ofneutrophil, lymphocyte, monocyte, eosinophil, basophil, platelet,reticulocyte, nucleated red blood cell (RBC), blast, promyelocyte,myelocyte, metamyelocyte, red blood cell (RBC), platelet, cell,bacteria, particulate matter, cell clump, or cellular fragment orcomponent. For example, in some embodiments, the apparatus may be usedfor automated image based white blood cell (WBC) differential counting,as well as automated identification of morphological abnormalitiesuseful in determining, diagnosing, prognosing, predicting, and/orsupporting a diagnosis of whether a subject is healthy or has a disease,condition, or infection and/or is responsive or non-responsive totreatment.

The above described and many other features and attendant advantages ofembodiments of the present invention will become apparent and furtherunderstood by reference to the following detailed description whenconsidered in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration, partly in section and not to scale,showing operational aspects of an exemplary flowcell, autofocus systemand high optical resolution imaging device for sample image analysisusing digital image processing.

FIG. 2 is a perspective illustration of a flowcell according to anexemplary embodiment.

FIG. 3 is a longitudinal median section view along lines 3-3 of theflowcell shown in FIG. 2.

FIGS. 3A and 3B provide additional section views of flowcells accordingto embodiments of the present invention.

FIG. 4 depicts aspects of an analyzer system according to embodiments ofthe present invention.

FIGS. 4A, 4B-1, and 4B-2 depict aspects of flowcells according toembodiments of the present invention.

FIGS. 4A-1 and 4A-2 depict cross-section views of sheath fluid (e.g.PIOAL) envelope and sample fluidstream dimensions within a flowcell at acannula exit port and an image capture site, respectively, according toembodiments of the present invention.

FIGS. 4C-4G, and 4D-1 depict aspects of cannula configurations accordingto embodiments of the present invention.

FIGS. 4H, 4I, and 4J depict aspects of results obtained using sheathfluid compositions, methods, and/or systems according to embodiments ofthe present invention.

FIGS. 4K and 4L depict aspects of sheath fluid and sample flow within aflowcell at an image capture site, according to embodiments of thepresent invention.

FIGS. 4K-1, 4K-2, and 4K-3 depict aspects of sheath fluid and sampleflow within a flowcell at an image capture site according to embodimentsof the present invention.

FIG. 4L-1 depicts aspects of fluid flow velocity within a flowcellaccording to embodiments of the present invention.

FIGS. 4M and 4N depict aspects of intracellular alignment and imaging,according to embodiments of the present invention.

FIG. 4O depicts aspects of the effect of PIOAL on particle and/orintracellular particle alignment and imaging according to embodiments ofthe present invention. In this comparison of images obtained using PIOALversus images obtained using a non PIOAL sheath fluid, it can be seenthat use of the PIOAL resulted in more in-focus cellular contents suchas lobes, cytoplasm, and/or granule.

FIGS. 4P and 4Q show comparison of images obtained using PIOAL versusimages obtained using standard sheath fluid. It can be seen that use ofPIOAL resulted in an improved RBC alignment.

FIG. 4R illustrates certain particle alignment results obtains usingflowcell configurations and sheath fluid compositions according toembodiments of the present invention.

FIGS. 5A and 5B illustrate aspects of sheath fluid and sample fluid flowcharacteristics, according to embodiments of the present invention.

FIG. 6 depicts aspects of particle imaging methods according toembodiments of the present invention.

FIGS. 7 and 8 depict aspects of flowstream strain rate according toembodiments of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to apparatus, systems, compositions, andmethods for analyzing a sample containing particles. In one embodiment,the invention relates to an automated particle imaging system whichcomprises an analyzer which may be, for example, a visual analyzer. Insome embodiments, the visual analyzer may further comprise a processorto facilitate automated analysis of the images.

According to this disclosure, a system comprising a visual analyzer isprovided for obtaining images of a sample comprising particles suspendedin a liquid. The system may be useful, for example, in characterizingparticles in biological fluids, such as detecting and quantifyingerythrocytes, reticulocytes, nucleated red blood cells, platelets, andwhite blood cells, including white blood cell differential counting,categorization and subcategorization and analysis. Other similar usessuch as characterizing blood cells from other fluids are alsoencompassed by embodiments of the present invention. Typically, theblood fluid sample is introduced into a flowing sheath fluid, and thecombined sheath and sample fluids are compressed with a narrowingflowpath transition zone that reduces the thickness of the sample ribbonfluid flow. Hence, particles such as cells can be oriented and/orcompressed within the blood fluid sample by the surrounding viscoussheath fluid, for example in combination with a geometric focusingeffect provided by a narrowing transition zone. Similarly, internalfeatures within blood cells can be aligned an oriented as a result of aviscosity differential between the sample fluid and the sheath fluid,for example in combination with a geometric focusing effect provided bya narrowing transition zone.

To facilitate the capacity, speed and effectiveness by which particlessuch as cells are categorized and/or subcategorized, it is advantageousto provide clear high quality images of the blood cells for automatedanalysis by the data processing system. According to the presentdisclosure, a prepared sample stream is arranged in a thin ribbon havinga stable position between opposite walls of a flowcell. The positioningof the sample stream and its flattening into a thin ribbon shape may beachieved by flow between layers of a PIOAL introduced into the flowcellthat differs in viscosity from the sample fluid and is flowed through asymmetrical narrowing transition zone of a flow channel.

Hematology—Particle Analysis System

Turning now to the drawings, FIG. 1 schematically shows an exemplaryflowcell 22 for conveying a sample fluid through a viewing zone 23 of ahigh optical resolution imaging device 24 in a configuration for imagingmicroscopic particles in a sample flow stream 32 using digital imageprocessing. Flowcell 22 is coupled to a source 25 of sample fluid whichmay have been subjected to processing, such as contact with a particlecontrast agent composition and heating. Flowcell 22 is also coupled toone or more sources 27 of a particle and/or intracellular organellealignment liquid (PIOAL), such as a clear glycerol solution having aviscosity that is greater than the viscosity of the sample fluid.

The sample fluid is injected through a flattened opening at a distal end28 of a sample feed tube 29, and into the interior of the flowcell 22 ata point where the PIOAL flow has been substantially establishedresulting in a stable and symmetric laminar flow of the PIOAL above andbelow (or on opposing sides of) the ribbon-shaped sample stream. Thesample and PIOAL streams may be supplied by precision metering pumpsthat move the PIOAL with the injected sample fluid along a flowpath thatnarrows substantially. The PIOAL envelopes and compresses the samplefluid in the zone 21 where the flowpath narrows. Hence, the decrease inflowpath thickness at zone 21 can contribute to a geometric focusing ofthe sample stream 32. The sample fluid ribbon 32 is enveloped andcarried along with the PIOAL downstream of the narrowing zone 21,passing in front of, or otherwise through the viewing zone 23 of, thehigh optical resolution imaging device 24 where images are collected,for example, using a CCD 48. Processor 18 can receive, as input, pixeldata from CCD 48. The sample fluid ribbon flows together with the PIOALto a discharge 33.

As shown here, the narrowing zone 21 can have a proximal flowpathportion 21 a having a proximal thickness PT and a distal flowpathportion 21 b having a distal thickness DT, such that distal thickness DTis less than proximal thickness PT. The sample fluid can therefore beinjected through the distal end 28 of sample tube 29 at a location thatis distal to the proximal portion 21 a and proximal to the distalportion 21 b. Hence, the sample fluid can enter the PIOAL envelope asthe PIOAL stream is compressed by the zone 21.

The digital high optical resolution imaging device 24 with objectivelens 46 is directed along an optical axis that intersects theribbon-shaped sample stream 32. The relative distance between theobjective 46 and the flowcell 33 is variable by operation of a motordrive 54, for resolving and collecting a focused digitized image on aphotosensor array.

According to some embodiments, the system can operate to hydrofocus thesample fluid ribbon 32. The term hydrofocus or hydrofocusing can referto a focusing effect which is influenced by a viscosity differencebetween the sheath and sample fluids, a geometric narrowing transitionzone of the flowcell, and a velocity difference between the sheath andsample fluids. Hydrodynamic flow results from the velocity differencebetween the sample and sheath fluid streams, which affects the flowribbon thickness and shape.

Flowcell

A practical embodiment of flowcell 22 is further depicted in FIGS. 2 and3. As shown here, flowcell 22 can be coupled with a sample source 25 andalso to a source 27 of PIOAL material. The sample fluid is injected intothe flowcell 22 via the cannula 29, for example through a distal exitport 31 of the cannula 29. Typically, the PIOAL sheath fluid is not in alaminar flow state as it travels through a curved channel section 41 inthe flowcell from the source 27 toward the viewing zone 23. However, theflowcell 22 can be configured so that the PIOAL sheath fluid is orbecomes laminar, or presents a flat velocity profile, as it flows pastthe distal exit port 31 where sample fluid is introduced into theflowing sheath fluid. The sample fluid and the PIOAL can flow along theflowcell 22 in a direction generally indicated by arrow A, and then outof the flowcell 22 via discharge 33. The flowcell 22 defines an internalflowpath 20 that narrows symmetrically (e.g. at transition zone 21) inthe flow direction A. The symmetry of the flowpath contributes to arobust and centered flow of the sample stream. The flowcell 22 isconfigured to direct a flow 32 of the sample enveloped with the PIOALthrough a viewing zone 23 in the flowcell, namely behind viewing port57. Associated with the viewport 57 is an autofocus pattern 44. Flowcell22 also has a rounded or recessed seat 58 which is configured to acceptor receive a microscope objective (not shown).

According to some embodiments, the autofocus pattern 44 can have aposition that is fixed relative to the flowcell 22, and that is locatedat a displacement distance from the plane of the ribbon-shaped samplestream 32. In the embodiment shown here, the autofocus pattern (target44) is applied directly to the flowcell 22 at a location that is visiblein an image collected through viewport 57 by a high optical resolutionimaging device (not shown). Flowcell 22 can be constructed from a singlepiece of material. Alternatively, flowcell 22 can be constructed of afirst or upper section or layer 22 a and a second or lower section orlayer 22 b. As shown here, a glass or transparent window pane 60 isattached to or integral with the first section 22 a. The pane 60 candefine at least a portion of the sample flowpath within the flowcell.Light from light source 42 can travel through an aperture or passage ofthe autofocus pattern 44 so as to illuminate sample particles flowingwithin the flow stream 32.

In some cases, the thickness of pane 60 can have a value within a rangefrom about 150 μm to about 170 μm. As noted above, the pane 60 candefine or form part of the flowpath or sheath (e.g. PIOAL) channel. Byusing a thin pane 60, it is possible to place the microscope objectivevery close to the sample fluid ribbon, and hence obtain highly magnifiedimages of particles flowing along the flowpath.

FIG. 3A depicts aspects of a flowcell embodiment, where a distancebetween the imaging axis 355 and the distal transition zone portion 316is about 8.24 mm. A distance between the distal transition zone portion316 and the cannula exit port 331 is about 12.54 mm. A distance betweenthe cannula exit port 331 and the sheath fluid entrance 301 is about12.7 mm. A distance between the cannula exit port 331 and a proximaltransition zone portion 318 is about 0.73 mm. FIG. 3B depicts aspects ofa flowcell embodiment where the cannula exit port has been moved to amore distal location relative transition zone, as compared to the FIG.3A embodiment. As shown here, the cannula distal end is advanced intothe narrowing transition zone of the flowcell, and a distance betweenthe imaging axis 355 and the distal transition zone portion 316 iswithin a range from about 16 mm to about 26 mm. In some case, thedistance between the imaging axis 355 and the distal transition zoneportion 316 is about 21 mm.

With returning reference to FIG. 1, the flowcell internal contour (e.g.at transition zone 21) and the PIOAL and sample flow rates can beadjusted such that the sample is formed into a ribbon shaped stream 32.The stream can be approximately as thin as or even thinner than theparticles that are enveloped in the ribbon-shaped sample stream. Whiteblood cells may have a diameter around 10 for example. By providing aribbon-shaped sample stream with a thickness less than 10 the cells maybe oriented when the ribbon-shaped sample stream is stretched by thesheath fluid, or PIOAL. Surprisingly stretching of the ribbon-shapedsample stream along a narrowing flowpath within PIOAL layers ofdifferent viscosity than the ribbon-shaped sample stream, such as higherviscosity, advantageously tends to align non-spherical particles in aplane substantially parallel to the flow direction, and apply forces onthe cells, improving the in-focus contents of intracellular structuresof cells. The optical axis of the high optical resolution imaging device24 is substantially normal (perpendicular) to the plane of theribbon-shaped sample stream. The linear velocity of the ribbon-shapedsample stream at the point of imaging may be, for example, 20-200mm/second. In some embodiments, the linear velocity of the ribbon-shapedsample stream may be, for example, 50-150 mm/second.

The ribbon-shaped sample stream thickness can be affected by therelative viscosities and flow rates of the sample fluid and the PIOAL.The source 25 of the sample and/or the source 27 of the PIOAL, forexample comprising precision displacement pumps, can be configured toprovide the sample and/or the PIOAL at controllable flow rates foroptimizing the dimensions of the ribbon-shaped sample stream 32, namelyas a thin ribbon at least as wide as the field of view of the highoptical resolution imaging device 24.

In one embodiment, the source 27 of the PIOAL is configured to providethe PIOAL at a predetermined viscosity. That viscosity may be differentthan the viscosity of the sample, and can be higher than the viscosityof the sample. The viscosity and density of the PIOAL, the viscosity ofthe sample material, the flow rate of the PIOAL and the flow rate of thesample material are coordinated to maintain the ribbon-shaped samplestream at the displacement distance from the autofocus pattern, and withpredetermined dimensional characteristics, such as an advantageousribbon-shaped sample stream thickness.

In a practical embodiment, the PIOAL has a higher linear velocity thanthe sample and a higher viscosity than the sample, thereby stretchingthe sample into the flat ribbon. The PIOAL viscosity can be up to 10centipoise.

Referring also to FIGS. 2 and 3, the internal flowpath of the flowcellnarrows downstream of the point of injection of the ribbon-shaped samplestream into the PIOAL, to produce a ribbon-shaped sample streamthickness, for example, up to 7 μm, and/or the internal flowpathproduces a ribbon-shaped sample stream width of 500-3,000 μm. Inexemplary embodiments, as depicted in FIG. 1, the internal flowpath ofthe flowcell begins a narrowing transition zone upstream of the point ofinjection of the sample stream into the PIOAL.

In another embodiment the internal flowpath narrows to produce aribbon-shaped sample stream thickness of 2-4 μm in thickness, and/or theinternal flowpath results in the ribbon-shaped sample stream of 2000 μmin width. These dimensions are particularly useful for hematology. Thethickness of the stream in this case is less than the diameter of someparticles, such as red blood cells in their relaxed state. Accordingly,those particles can become reoriented to face their wider a dimension tothe imaging axis, which is helpful in revealing distinguishingcharacteristics.

The linear velocity of the ribbon-shaped sample stream can be limitedsufficiently to prevent motion blurring of the digitized image at theimage exposure time of the photosensor array. The light source canoptionally be a strobe light that is flashed to apply high incidentamplitude for a brief time. Inasmuch as the autofocus pattern 44 and theimage are in the same field of view, the light source is configured toilluminate the ribbon-shaped sample stream and the autofocus patternsimultaneously. However in other embodiments, the field of view forimaging and for autofocus can be different, e.g., illuminated and/orimaged separately.

The subject developments have method as well as apparatus aspects. Amethod of focusing a visual analyzer comprises focusing a high opticalresolution imaging device 24, which may be a digital high opticalresolution imaging device or the digital image capture device, on anautofocus pattern 44 fixed relative to a flowcell 22, wherein theautofocus pattern 44 is located at a displacement distance 52 from aribbon-shaped sample stream 32. The digital high optical resolutionimaging device 24 has an objective with an optical axis that intersectsthe ribbon-shaped sample stream 32. A relative distance between theobjective and the flowcell 22 is varied by operation of a motor drive54, whereas the distance along the optical axis between the high opticalresolution imaging device and the point of optimal focus is known. Thedigital high optical resolution imaging device is configured to resolveand collect a digitized image on a photosensor array. The motor drive isoperated to focus on the autofocus pattern in an autofocus process. Themotor drive then is operated over the displacement distance, therebyfocusing the high optical resolution imaging device on the ribbon-shapedsample stream.

The method further can further include forming the ribbon-shaped samplestream into a ribbon-shape. The ribbon shape is presented such that theoptical axis of the high optical resolution imaging device issubstantially perpendicular to the ribbon-shaped sample stream, namelynormal to the plane of the ribbon-shaped stream.

FIG. 4 depicts aspects of a system 400 for imaging particles in a bloodfluid sample. As shown here, system 400 includes a sample fluidinjection system 410, a flowcell 420, and image capture device 430, anda processor 440. The flowcell 420 provides a flowpath 422 that transmitsa flow of the sheath fluid, optionally in combination with the samplefluid. According to some embodiments, the sample fluid injection system410 can include or be coupled with a cannula or tube 412. The samplefluid injection system 410 can be in fluid communication with theflowpath 422 (e.g. via sample fluid entrance 402), and can operate toinject sample fluid 424 through a distal exit port 413 of the cannula412 and into a flowing sheath fluid 426 within the flowcell 420 so as toprovide a sample fluid stream 428. For example, the processor 440 mayinclude or be in operative association with a storage medium having acomputer application that, when executed by the processor, is configuredto cause the sample fluid injection system 410 to inject sample fluid424 into the flowing sheath fluid 426. As shown here, sheath fluid 426can be introduced into the flowcell 420 by a sheath fluid injectionsystem 450 (e.g. via sheath fluid entrance 401). For example, theprocessor 440 may include or be in operative association with a storagemedium having a computer application that, when executed by theprocessor, is configured to cause the sheath fluid injection system 450to inject sheath fluid 426 into the flowcell 420. As depicted in FIG. 4,the distal exit port 413 of cannula 412 can be positioned at a centrallocation along the length of the narrowing transition zone 419. In somecases, the distal exit port can be positioned more closely to thebeginning (proximal portion) of the transition zone 419. In some cases,the distal exit port can be positioned more closely to the end (distalportion) of the transition zone 419. In some cases, the distal exit port413 can be positioned entirely outside of the transition zone 419, forexample as depicted in FIG. 3A (where distal exit port 331 is disposedproximal to the narrowing transition zone).

The sample fluid stream 428 has a first thickness T1 adjacent theinjection tube 412. The flowpath 422 of the flowcell having a decreasein flowpath size such that the thickness of the sample fluid stream 428decreases from the initial thickness T1 to a second thickness T2adjacent an image capture site 432. The image capture device 430 isaligned with the image capture site 432 so as to image a first pluralityof the particles from the first sample fluid at the image capture site432 of the flowcell 420.

The processor 440 is coupled with the sample fluid injector system 410,the image capture device 430, and optionally the sheath fluid injectionsystem 450. The processor 440 is configured to terminate injection ofthe first sample fluid into the flowing sheath fluid 426 and begininjection of the second sample fluid into the flowing sheath fluid 426such that sample fluid transients are initiated. For example, theprocessor 440 may include or be in operative association with a storagemedium having a computer application that, when executed by theprocessor, is configured to cause the sample fluid injection system 410to inject the second sample fluid into the flowing sheath fluid 426 suchthat sample fluid transients are initiated.

Further, the processor 440 is configured to initiate capture of an imagea second plurality of the particles from the second sample fluid at theimage capture site 432 of the flowcell 420 after the sample fluidtransients and within 4 seconds of the imaging of the first pluralitythe particles. For example, the processor 440 may include or be inoperative association with a storage medium having a computerapplication that, when executed by the processor, is configured to causethe image capture device 430 to initiate capture of an image a secondplurality of the particles from the second sample fluid at the imagecapture site 432 of the flowcell 420 after the sample fluid transientsand within four seconds of the imaging of the first plurality theparticles.

Accordingly, embodiments of the present invention encompass a system 400for imaging a plurality of particles in a blood fluid sample 424 havinga sample fluid viscosity The system 400 can be used with a sheath fluid426 having a sheath fluid viscosity that differs from the sample fluidviscosity by a viscosity difference in a predetermined viscositydifference range. The system 400 can include a flowcell 420 having aflowpath 422 and a sample fluid injection tube 412. The flowpath 422 canhave a reduction in flowpath size or narrowing transition zone. Further,the system 400 can include a sheath fluid input 401 in fluidcommunication with the flowpath 422 of the flowcell 420 so as totransmit a flow of the sheath fluid along the flowpath 422 of theflowcell 420. The system 400 can also include a blood fluid sample input402 in fluid communication with the injection tube 412 of the flowcell420 so as to inject a flow or stream 428 of the blood fluid sample intothe flowing sheath fluid 428 within the flowcell 420. For example, thesample fluid 424 can exit the distal exit port 423 of the cannula 412and into an envelope of the flowing sheath fluid 426 to form a sampleribbon 428 therein.

As the sheath fluid 426, along with the sample fluid ribbon 428 formedfrom the sample fluid 424, flow through a reduction 419 in flowpath sizeand toward an imaging site 432, a viscosity hydrofocusing effect inducedby an interaction between the sheath fluid 426 and the sample fluid 424associated with the viscosity difference, in combination with ageometric hydrofocusing effect induced by an interaction between thesheath fluid 426 and the sample fluid 424 associated with the reductionin flowpath size, provides a target imaging state in at least some ofthe plurality of particles at the imaging site 432. As shown here, thesystem 400 also includes an imaging device 430 that images the pluralityof particles at the imaging site 432.

As shown in the flowcell embodiment depicted in FIG. 4A, a decrease inflowpath size (e.g. at transition zone 419 a) can be defined by opposedwalls 421 a, 423 a of the flowpath 422 a. The opposed walls 421 a, 423 acan angle radially inward along the flowpath 422 a, generally symmetricabout a transverse plane 451 a that bisects the sample fluid stream 428a. The plane 451 a can bisect the sample stream 428 a where the samplestream has a first thickness T1, at a location where the sample stream428 a exits a distal portion 427 a of the cannula or sample injectiontube 412 a. Similarly, the plane 451 a can bisect the sample stream 428a where the sample stream has a second thickness T2, at a location wherethe sample stream 428 a passes the image capture site 432 a. Accordingto some embodiments, the first thickness T1 has a value of about 150 μmand the second thickness T2 has a value of about 2 μm. In such cases,the compression ratio of the sample ribbon stream is 75:1. According tosome embodiments, the first thickness T1 has a value within a range fromabout 50 μm to about 250 μm and the second thickness T2 has a valuewithin a range from about 2 μm to about 10 μm. As the sample streamfluid flows through the flowcell, the ribbon thins out as it acceleratesand is stretched. Two features of the flowcell can contribute tothinning of the sample fluid ribbon. First, a velocity differencebetween the sheath fluid envelope and the sample fluid ribbon canoperate to reduce the thickness of the ribbon. Second, the taperedgeometry of the transition zone can operate to reduce the thickness ofthe ribbon. As depicted in FIG. 4A, the distal exit port 413 a ofcannula 412 a can be positioned at a central location along the lengthof the narrowing transition zone 419 a. In some cases, the distal exitport can be positioned more closely to the beginning (proximal portion415 a) of the transition zone 419 a. In some cases, the distal exit portcan be positioned more closely to the end (distal portion 416 a) of thetransition zone 419 a. In some cases, the distal exit port 413 a can bepositioned entirely outside of the transition zone 419 a, for example asdepicted in FIG. 3A (where distal exit port 331 is disposed proximal tothe narrowing transition zone).

As depicted in FIG. 4A (as well as in FIGS. 4 and 4B-1), the transitionzone 419 a can be defined by an angular transitions at the proximal (415a) and distal (416 a) portions. It is also understood that thetransition zone 419 a can instead present smooth or curved transitionsat the proximal (415 a) and distal (416 a) portions, similar to thesmooth or curved transitions as depicted in FIGS. 1, 3, 3A, 3B, and4B-2).

Typically, the first thickness T1 is much larger than the size of thesample particles, and hence the particles are contained entirely withinthe sample ribbon stream. However, the second thickness T2 may besmaller than the size of certain sample particles, and hence thoseparticles may extend out of the sample fluid and into surrounding sheathfluid. As shown in FIG. 4A, the sample ribbon stream can flow generallyalong the same plane as it exits the cannula and travels toward theimage capture site.

The flowcell can also provide a separation distance 430 a between thedistal cannula portion 427 a and the image capture site 432 a. Accordingto some embodiments, the distal portion 427 a of the sample fluidinjection tube 412 a can be positioned at an axial separation distance430 a from the image capture site 432 a, where the axial separationdistance 432 a has a value of about 21 mm. In some cases, the axialseparation distance 430 a has a value within a range from about 16 mm toabout 26 mm.

The axial separation distance 430 a between the cannula exit port andimage capture site can impact the transition time for the sample fluidas the fluid travels from the exit port to the image capture site. Forinstance, a relatively shorter axial separation distance 430 a cancontribute to a shorter transition time, and a relatively longer axialseparation distance 430 a can contribute to a longer transition time.

The position of the exit port at the cannula distal portion 427 arelative to the flowpath transition zone 419 a, or relative to theproximal portion 415 a of the flowpath transition zone 419 a, can alsoinference the transition time for the sample fluid as the fluid travelsfrom the exit port to the image capture site. For example, the sheathfluid may have a relatively slower speed at the proximal portion 415 a,and a relatively faster speed at a location between the proximal portion415 a and the distal portion 416 a. Hence, if the cannula exit port atdistal portion 427 a is positioned at the proximal portion 415 a, itwill take a longer amount of time for the sample fluid to reach theimage capture site, not only because the travel distance is longer, butalso because the initial speed of the sample fluid after it exits thecannula distal port is slower (due to the slower sheath fluid speed).Put another way, the longer the sample fluid is present in the thickerportion (e.g. near proximal portion 415 a) of the flowcell, the longerit takes the sample to reach the image capture site. Conversely, if thecannula exit port at distal portion 427 a is positioned distal to theproximal portion 415 a (e.g. at a central location between proximalportion 415 a and distal portion 416 a, as depicted in FIG. 4A), it willtake a shorter amount of time for the sample fluid to reach the imagecapture site, not only because the travel distance is shorter, but alsobecause the initial speed of the sample fluid after it exits the cannuladistal port is faster (due to the faster sheath fluid speed). Asdiscussed elsewhere herein, the sheath fluid is accelerated as it flowsthrough the transition zone 419 a, due to the narrowing cross-sectionalarea of the zone 419 a.

According to some embodiments, with a shorter transition time, more timeis available for image collection at the image capture site. Forexample, as the duration of the transition time from the cannula distaltip to the imaging area decreases, it is possible to process moresamples in a specific amount of time, and relatedly it is possible toobtain more images in a specific amount of time (e.g. images perminute).

Although there are advantages associated with positioning the exit portof the cannula distal portion 427 a more closely to the image capturesite 432 a, it is also desirable to maintain a certain distance betweenthe port and the capture site. For example, as depicted in FIG. 3, anoptical objective or front lens of an imaging device can be positionedin the seat 58 of the flowcell 22. If the exit port 31 of the cannula istoo close to the seat 58, then the sample fluid may not be sufficientstabilized after it is injected into the sheath fluid so as to providedesired imaging properties at the image capture site. Similarly, it maybe desirable to maintain the tapered transition region 21 at a distancefrom the viewing zone 23, so that the tapered region does not interferewith the positioning of the seat 58 which receives the image capturedevice objective.

With continuing reference to FIG. 4A, the downstream end 427 a of thesample fluid injection tube 412 a can be positioned distal to a proximalportion 415 a of the flowpath transition zone 419 a. Relatedly, thedownstream end 427 a of the sample fluid injection tube 412 a can bepositioned proximal to a distal portion 416 a of the flowpath transitionzone 419 a. Hence, according to some embodiments, the sample fluid canbe injected from the injection cannula 412 a and into the flowcell at alocation within the transition zone 419 a.

According to some embodiments, symmetry in the decrease in flowpath size(e.g. at flowpath transition zone 419 a) operates to limit particlemisalignment in the blood fluid sample. For example, such symmetry canbe effective to limit red blood cells imaging orientation misalignmentin the blood fluid sample to less than about 20%.

According to some embodiments, methods disclosed herein are operable tothe flagging rate during blood count analysis to below 30%, 29%, 28%,27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,13%, 12%, 11%, 10%, 9%, 8%, 7%, 6% or 5% of samples.

According to some embodiments, the image capture site 432 a has a fieldof view 433 a of between about 150 μm×150 μm and 400 μm×400 μm. In somecases, the image capture site 432 a has a field of view 433 a of about275 μm×275 μm. In some cases, the field of view can be defined in termsof length times width. If expressed as surface area, a 275 μm×275 μmfield of view has an area of 75,625 μm². According to some embodiments,the field of view can be determined by the imaging device objective andits magnification. In some cases, the field of view can correspond tothe extent of the field (area) that is imaged by the collection optics(e.g. objective, tube lens, and camera). In some cases, the field ofview is much smaller than the viewing port of transparent area at theimage capture site.

FIGS. 4A-1 and 4A-2 illustrate the effects of hydrofocusing on thesample stream as it travels from the cannula exit port to the imagecapture site. As shown in FIG. 4A-1, the sample stream can have a heightH(S) of about 150 μm and a width W(S) of about 1350 μm. Further, thePIOAL sheath stream can have a height H(P) of about 6000 μm and a widthW(P) of about 4000 μm. Subsequent to the hydrofocusing, as shown in FIG.4A-2, the sample stream can have a height H(S) of about 2 μm and a widthW(S) of about 1350 μm. Further, the PIOAL sheath stream can have aheight H(P) of about 150 μm and a width W(P) of about 4000 μm. In oneembodiment, the cross sectional area of the PIOAL sheath stream at thecannula exit is 40 times larger than the cross sectional area near theimage capture site.

According to some embodiments, it can be useful to determine thecross-section of the flowcell channel at the image capture site. Thiscan correspond to the PIOAL sheath stream height H(P) of about 150 μmand a width W(P) of about 4000 μm as depicted in FIG. 4A-2. It can alsobe useful to determine the volumetric flow rate of the combined sampleand sheath fluid streaming through the flowcell at the image capturesite. When the cross-section area and the flow rate are known, it ispossible to determine the velocity of the combined sample and sheathfluid at the image capture site.

According to some embodiments, the flow of the sample and sheath fluidsthrough the flowcell can be approximated with a parallel plate profilemodel. Relatedly, the flow rate in the center of the sample fluid stream(e.g. as depicted in FIG. 4A-2), can be about 1.5 times the average flowrate of the combined sample and sheath fluid stream.

According to some embodiments, the cross-sectional area of the sampleflow at the cannula exit (e.g. W(S)×H(S) in FIG. 4A-1) is 40 timeslarger than the cross-sectional area of the sample flow at the imagingsite (e.g. W(S)×H(S) in FIG. 4A-2). The volumetric flow rate of sheathfluid at the imaging area can be about 45 μL/second. The volumetric flowrate of sample fluid at the imaging area can be about 0.232 μL/second.In some cases, the cross-sectional area of the combined sheath andsample streams at the imaging site is 600,000 μm². In some cases, theaverage flowstream velocity at the imaging site is 75 mm/second.

The flow rate or velocity can be determined as the rate that results inclear and focused cellular images. Exemplary flow rates and velocitieswere discovered based on flow rates of the two samples that wereobserved to achieve certain sample flowstream ribbon shapes orcharacteristics at the imaging site. For example, at flow rate of about75 mm/sec (or within a range from 20-200 mm/sec), the cells do not flowtoo slow such that there are overlaps of cells in consecutive images,and the cells do not flow too fast such that ghosting effects arecreated (blurred image). Relatedly, by avoiding excessively high flowrates, it is possible to conserve more reagent and sample. According tosome embodiments, an optimal or desired linear velocity can be achievedby either changing the volumetric flow (pump rate) or the shape ofcannula.

The flow velocity of the sample stream through the image capture zonecan also be related to the performance of the image capture devicerelative to the flowcell function. For example, if the sample stream ifflowing too quickly, it may be difficult to obtain clear images ofparticles contained in the sample (e.g. the shutter speed of the imagecapture device may be too low, thus producing a blurred image).Similarly, if the sample stream is flowing too slowly, the image capturedevice may obtain consecutive images of the same particle (e.g. the sameparticle remains in the capture frame during two image captures). Insome embodiments, the velocity of the sample ribbon can be modulated(e.g. by adjusting any of a variety of the flowcell operationalparameters) relative to the image capture rate, so that there is minimalflow between frame captures, and hence a high percentage of the sampleis imaged.

According to some embodiments, the particle analysis system andassociated components can be configured so that as the sheath fluid andfluid sample flow through the flowcell, the sheath fluid can flow at asheath fluid volumetric rate of 45 μL/s and the fluid sample can flow ata fluid sample volumetric flow rate of 0.232 μL/s (or within a rangefrom 0.2 to 0.35 μL/s). In some cases, the ratio of the sheath fluidflow rate to the sample fluid flow rate is about 200. In some cases, theratio of the sheath fluid flow rate to the sample fluid flow rate has avalue within a range from about 70 to 200. In some cases, the ratio ofthe sheath fluid flow rate to the sample fluid flow rate is about 193.In some cases, the ratio of the sheath fluid flow rate to the samplefluid flow rate is about 70. In some instances, a ratio of sheath fluidvolume to fluid sample volume flowing within the flowcell can be withina range from 25:1 to 250:1.

According to some embodiments, the system and associated components canbe configured so that as sheath fluid and fluid sample flow through theflowcell 420, the sheath fluid can flow at a sheath fluid velocity of 75mm/sec before the imaging area and the fluid sample can flow at a fluidsample velocity of 130 mm/sec before the imaging area. In someinstances, a ratio of sheath fluid volume to fluid sample volume flowingwithin the flowcell can be within a range from 100:1 to 200:1.

In some instances, a flowcell can have a minimum compression ratio ofabout 50:1 and a maximum compression ratio of about 125:1. In somecases, the minimum compression ratio can be about 30:1 or 20:1. Thiscompression ratio refers to the ratio of flow stream thicknessesH(S):H(S) when comparing FIG. 4A-1 to FIG. 4A-2. This compression ratiocan be influenced by a combination of geometric compression (e.g. theratio of the sheath fluid thicknesses H(P):H(P) when comparing FIG. 4A-1to FIG. 4A-2, which can also correspond generally to the dimensions ofthe flowcell narrowing tapered transition zone 419 a shown in FIG. 4A)and a hydrodynamic compression (e.g. also corresponding to a differencein velocity). According to some embodiments, the geometric compressionratio is about 40:1.

The decrease in flowpath size, corresponding to the transition zone, canbe defined by a proximal flowpath portion having a proximal thickness orheight, and a distal flowpath portion having a distal thickness orheight that is less than the proximal thickness or height. For example,as shown in the partial views of FIGS. 4B-1 and 4B-2, the transitionzone 419 b of the flowpath can have a length L between a proximalportion 415 b and a distal portion 416 b, where the proximal portion 415b has a proximal height 417 b, and the distal portion 416 b has a distalheight 418 b. As depicted in FIG. 4B-2, and as noted elsewhere herein,the shape or contour of the transition zone can be curved or smooth, andfor example can be provided in the shape of an S-curve, a sigmoidalcurve, or a tangent curve. According to some embodiments, the proximalheight 417 b has a value of about 6000 μm. In some cases, the proximalheight 417 b has a value within a range from about 3000 μm to about 8000μm. According to some embodiments, the distal height 418 b has a valueof about 150 μm. In some cases, the distal height 418 b has a valuewithin a range from about 50 μm to about 400 μm.

The geometry of the transition zone 419 a can provide a first angle α1between the first flowpath boundary 403 b and the bisecting transverseplane 451 b, and a second angle α2 between the second flowpath boundary404 b and the bisecting transverse plane 451 b. In some cases, angle α1is about 45 degrees and angle α2 is about 45 degrees. In some cases,angle α1 has a value within a range from about 10 degrees to about 60degrees. In some cases, angle α2 has a value within a range from about10 degrees to about 60 degrees. According to some embodiments, angles α1and α2 have the same value. The angles α1 and α2 can be selected so asto maintain laminar flow or minimize turbulence of the sample fluid asit travels from proximal portion 415 b to distal portion 416 b, which inturn can enhance alignment of particles within the sample along thetransverse plane 451 b. As noted above with reference to FIG. 4A, thedistal and proximal boundaries or portions of the transition zone may becurved or smooth, instead of angled.

FIG. 4C depicts features of an exemplary cannula or sample feed tube 400c according to embodiments of the present invention, where the cannulahas a length L. FIG. 4D depicts a longitudinal cross-section of cannula400 d. As shown here, the cannula 400 d includes a distal flattenedsection 410 d, a central tapered section 420 d, and a proximal tubularportion 430 d. As depicted in FIG. 4C-1, an exemplary cannula or samplefeed tube 400 c-1 can have a distal portion 410 c-1 and a proximalportion 430 c-1. In some cases, the distal portion 410 c-1 has a lengthof about 1.359 mm and a width of about 1.43 mm. In some cases, the exitport of the distal end has an exit width W(E) of about 1.359 mm.According to some embodiments, a cannula may have an internal flowpathgeometry that is different from what is depicted in FIGS. 4C and 4D. Forexample, as illustrated in FIG. 4D-1, the cannula 400 d-1 does notinclude a tapered central section having an expanded flow areacross-section. As depicted in FIG. 4D-1, cannula 400 d-1 has a distalsection 410 d-1, a central tapered section 420 d-1 having a taperinginner diameter, and a proximal section 430 d-1. Corresponding to thetapering inner diameter of central section 420 d-1, the cross-sectionalinner area of 410 d-1 is smaller than the cross-sectional inner area of430 d-1.

A hematology system according to embodiments of the present inventioncan process a blood sample having a volume of about 150 μL. Theaspirated blood volume can be about 120-150 μL. In some cases, theminimum available blood volume in the sample tube is about 500 μL for anautomatic sampling mode and about 250 μL for manual sampling mode. Thecannula or injection tube 400 d shown in FIG. 4D has an internal volumeof about 13 uL. According to some embodiments, the cannula or injectiontube has an internal volume of less than about 30 uL.

FIG. 4E illustrates a transverse cross-section of a distal flattenedsection 410 e. As shown here, the distal section 410 e has an innerwidth W(I) and an inner height H(I), through which a sample streamflows. Further, the distal section 410 e has an outer width W(O) and anouter height H(O). As depicted in FIGS. 4D and 4E taken in combination,the distal portion 410 e of the sample fluid injection tube has anoutlet port P having a height H(I) and a width W(I), where the heightH(I) is less than the width W(I). According to some embodiments, theheight H(I) of the outlet port P of distal portion 410 e (or the innerheight of the distal portion 410 d) can have a value of about 150 μm. Insome cases, the height H(I) can be within a range from about 50 μm toabout 250 μm. According to some embodiments, the width W(I) of theoutlet port P of distal portion 410 e (or the inner width of the distalportion 410 d) can have a value of about 1350 μm. In some cases, thewidth is about 1194 μm. In some cases, the width W(I) can have a valuewithin a range from about 500 μm to about 3000 μm. In some cases, distalflattened section 410 d can be manufactured by applying a clamping forceto a tube or conduit.

FIG. 4F illustrates a transverse cross-section of a central taperedsection 420 f. As shown here, the central tapered section 420 f has aninner diameter D(I) through which a sample stream flows. Further, thecentral tapered section 420 f has an outer diameter D(O). FIG. 4Gillustrates a transverse cross-section of a proximal section 430 g. Asshown here, the proximal section 430 g has an inner diameter D(I)through which a sample stream flows. Further, the distal section 430 ghas an outer diameter D(O).

As depicted in FIG. 4D, the injection tube or cannula 400 d can have aproximal portion 430 d having a first flow cross-section area (e.g.π*(D/2)² shown in FIG. 4G), a distal portion 410 d having a second flowcross-section area (e.g. W(I)*H(I) shown in FIG. 4E) that is less thanthe first flow cross-section area, and a third portion 420 d disposedbetween the proximal portion 430 d and the distal portion 410 d. Thethird portion 420 d can have a third flow cross-section (e.g. π*(D/2)²shown in FIG. 4F) that is larger than the first and second flowcross-sections. In some instance, the outer diameter D(O) of proximalportion 430 g is about 1067 μm and the inner diameter D(I) of proximalportion 430 g is about 813 μm.

Cellular Structure, Content, and Alignment

According to some embodiments, to accomplish staining and visualizationof white blood cells, it is helpful to lyse red blood cells in thesample and permeabilize the white blood cells so as to allow the stainto incorporate with the white blood cells. It is often desirable toobtain a stain of the white blood cells with little to no change inmorphology to the cells. Further, it is often desirable to obtainstaining properties which resemble a Wright stain. What is more, it isoften desirable to obtain a high red cell alignment (e.g. target >90%).

FIG. 4H (upper panel) depicts results obtained using a stain formulationthat does not include glutaraldehyde. It was observed that the cellsfell apart as a result of shear forces encountered in the flowcell.Although a good stain of the nucleus was achieved, the nucleus itselfappeared deformed, and the cell membrane appeared damaged. In sum, whenimaged the cell appeared to be destroyed due to disruption to thecellular content and structure.

FIG. 4H (lower panel) depicts WBC results obtained using a stainformulation that includes glutaraldehyde. As shown here, the cellmembranes are intact and the cells are round. Hence, it was observedthat the version of the stain which did not use glutaraldehyde (e.g.shown in FIG. 4H, upper panel) resulted in resulting in weakened WBC's.Although the WBC's are more intact in FIG. 4H (lower panel) the nucleusportions are damaged.

The sheath fluid (PIOAL) used to obtain the FIG. 4H (lower panel) imagesincluded 30% glycerol. In contrast, the sheath fluid (PIOAL) used toobtain the FIG. 4I (upper panel) images included 6.5% glycerol. Thelower concentration of glycerol resulted in a better morphology, withthe nucleus mostly unchanged. Hence, it was observed that the cellmembrane in FIG. 4I (upper panel) is even more intact that than the cellmembrane in FIG. 4H (lower panel). The lower glycerol concentration inFIG. 4I (upper panel) can operate to reduce the viscosity difference,thereby reducing the shear force. If excessive shear force is present,the force can destroy the cell membranes. The glycerol may have someproperties that are incompatible with the cells and thus a higherconcentration of glycerol may also destroy the cell membranes. Hence, itis possible to conclude that the damage to the nucleus depicted in FIG.4H (upper panel) can be the result of the 30% glycerol in the sheathfluid.

When the glycerol concentration was lowered to 6.5% as depicted in FIG.4I (upper panel), however, the alignment of the red blood cells in thesample fluid was observed to diminish.

Various alternative PIOAL formulations were used in an attempt to obtainimproved alignment in red blood cells, but these alternativeformulations did not provide satisfactory results. For example, severaldifferent viscosity enhancers were tried, but many of them exhibitedbehavior similar to that of the higher 30% glycerol formulation, suchthat the cell contents were damaged.

It was discovered that by using polyvinylpyrrolidone (PVP) and 5%glycerol as a viscosity agent component, it was possible to obtain asheath fluid having a viscosity that matched the viscosity of the 30%glycerol formulation (and hence improved alignment results wereachieved) without the negative effects of destroying the nucleus. FIG.4I (lower panel) depicts results obtained using a PIOAL with 5% glyceroland 1% PVP. Hence, it can be seen that the viscosity agent in the sheathfluid retains viability of cells in the sample fluid stream, leavingstructure and content of the cells intact, for example when cells flowthrough the flowcell and are exposed to the flowing sheath fluid.According to some embodiments, the concentration percentage of glycerolis expressed in terms of (v/v) and the concentration percentage of PVPis expressed in terms of (w/v).

FIG. 4J depicts image capture results based on a traditional microscopewet mount technique (left column) as compared to a flowcell techniqueaccording to embodiments of the present invention (right column). Thewet mount procedure can be considered as a target standard for imageclarity and quality. It was observed that techniques involving sheathfluids and flow cell designed as disclosed herein were effective inachieving image clarity and quality equivalent to that of the wet mountprocedure.

According to some embodiments, a flowstream ribbon can split when theviscosity differential between the sample fluid and the sheath fluidexceeds a certain threshold. According to some embodiments, a flowstreamribbon split was observed when using a sheath fluid containing glycerolat 60%.

As shown in FIG. 4K, a sample stream ribbon R flowing through an imagecapture site 432 k of a flowcell 420 k can have a thickness T of about 2μm. In some cases, thickness T of the sample stream ribbon can be up toabout 3 μm. Typically, cells or particles that are smaller than thesample stream thickness will be contained within the ribbon. Anexemplary red blood cell (RBC) can be present as a biconcave disk andcan have a diameter D of between about 6.2 μm and about 8.2 μm. Further,an exemplary red blood cell can have a maximum thickness T1 of betweenabout 2 μm and about 2.5 μm and a minimum thickness T2 of between about0.8 μm and about 1 μm. In some cases, red blood cells can have athickness of up to about 3 μm. Exemplary human platelets can vary insize, and can also have a thickness or diameter of about 2 μm. Althoughnot shown to scale here, the flowcell can define a flow path thickness Hhaving a value of about 150 μm, at the image capture site. In somecases, the flowpath thickness F has a value between 50 μm and 400 μm.This flowpath thickness F can also correspond to the distal height 418 bof distal portion 461 b depicted in FIGS. 4B-1 and 4B-2.

As shown in FIG. 4K, the ratio of the thickness T of the sample fluidstream to the thickness of the particle (red blood cell) is about 1:1.According so some embodiments, a ratio of the thickness T of the samplefluid stream at the image capture site to a size of one of the particlesis within a range from 0.25 to 25. In some cases, the thickness T canhave a value within a range from 0.5 μm to 5 μm. A viscositydifferential between the sheath fluid and the sample fluid can beselected so as to achieve a desired positioning of the ribbon samplestream within the flowcell.

Viscosity differences between fluid of the sample ribbon R and thesheath fluid can operate to align or orient particles in the samplestream, for example red blood cells, along the direction of the flow.When so aligned, as shown in FIG. 4K, the imaging device or camera canobtain images of the red blood cells such they appear round, because themajor surface of the blood cell is facing toward the camera. In thisway, the red blood cell assumes an alignment that presents a lowresistance relative to the flow. Hence, the relative viscositycharacteristics of the sheath fluid and the sample fluid can contributeto a high percentage or number of red blood cells facing toward thecamera, thus enhancing the evaluation capability of the particleanalysis system.

According to some embodiments, the viscosity characteristics of thesheath fluid operate to limit particle misalignment in the blood fluidsample. For example, viscosity differentials can be effective to limitred blood cells imaging orientation misalignment in the blood fluidsample to less than about 10%. That is, 90 or more red blood cells outof 100 red blood cells in a sample can be aligned so that their majorsurfaces face toward the imaging device. A symmetrical narrowingtransition zone can provide a value of 20%. As discussed elsewhereherein, for example with reference to FIG. 4R, it is possible to comparealignment results obtained from an analyzer configuration that involvesa symmetrical narrowing flowcell transition zone and a viscous sheathfluid to alignment results obtained from an analyzer configuration thatinvolves a symmetrical narrowing flowcell transition zone without theuse of a viscous sheath fluid. Use of a viscous sheath fluid can reducethe percentage of misaligned cells. According to some embodiments, thesheath fluid has an index of refraction similar to that of water (i.e.n=1.3330). In some cases, the sheath fluid has a water content of about89%. In addition to alignment effects observed as a result of theviscosity differential, alignment effects are also observed as a resultof a bilateral tapered transition zone. In some cases, it is observedthat a bilateral (i.e. symmetrical) tapered transition zone is twice aseffective at aligning particles as compared to an asymmetric taperedtransition zone design.

Efficient alignment of the red blood cells can contribute to improveddiagnosis. In some cases, the shape of the imaged red blood cells can beused to determine whether a patient from whom the sample is obtained hasa particular physiological condition or disease. For example, patientswith sickle cell disease present with blood cells having an abnormalshape (i.e. in the shape of a sickle). Hence, by obtaining high qualityimages of aligned red blood cells, it is possible to ensure an accuratediagnosis. Other shape variations in red blood cells, for example redblood cells having thin peripheral area and a large flat central area,whereby the red blood cell appears to have the profile of a bicycletire, can effectively be imaged using the instant alignment techniques.Similarly, red blood cells having a small central portion, and a thickperipheral area, whereby the red blood cell appears to have the profileof a truck tire, can be imaged for diagnostic purposes. The improvedimaging techniques disclosed herein are also useful for evaluating otherred blood cell characteristics, such as hemoglobin content, ironcontent, and the like.

Without being bound by any particular theory, it is believed that aviscosity differential between the viscosity of the sheath fluid and theviscosity of the sample fluid produces a modified parabolic profile,wherein the profile is generally parabolic and has a central bumpcorresponding to a center area of the flow where the acceleration isincreased, and the central bump contributes to alignment of sampleparticles or intraparticle organelles. According to some embodiments,the velocity difference between the sheath and sample ribbon and theviscosity difference generate shear forces to increase alignment of theorganelles or intracellular particles. Exemplary aspects of the sheathfluid parabolic profile are discussed in co-pending U.S. patentapplication Ser. No. 14/216,533, the content of which is incorporatedherein by reference.

White blood cells are typically larger than red blood cells andplatelets. For example, exemplary neutrophils and eosinophils can have adiameter of between about 10 μm and about 12 μm. Exemplary basophils canhave a diameter of between about 12 μm and about 15 μm. Exemplarylymphocytes (small) can have a diameter of between about 7 μm and about8 μm, and exemplary lymphocytes (large) can have a diameter of betweenabout 12 μm and about 15 μm. Exemplary monocytes can have a diameter ofbetween about 12 μm and about 20 μm. The configuration of the particleanalysis system, including interaction between the sheath fluid and thefluid sample ribbon as they pass through the flowcell, can operate tocompress white blood cells as they travel through the image capture site432 l, as indicated in FIG. 4L. Hence, for example, a central portion ofthe white blood cell (WBC) can be positioned within the sample fluidribbon R, and peripheral portions of the white blood cell can bepositioned within the sheath fluid. Hence, as the white blood cell istransported through the flowcell by the ribbon, the sides of the whiteblood cell can extend into the sheath fluid. The numerical values orranges for the thickness T of sample stream ribbon R, and the thicknessF of the flowpath as discussed above with regard to FIG. 4K aresimilarly applicable to FIG. 4L.

According to some embodiments, viscosity differences between the sheathfluid and the sample fluid can operate to align organelles or otherintracellular features which are present within cells such as whiteblood cells. Without being bound by any particular theory, it isbelieved that shear forces associated with the viscosity differentialbetween the sheath fluid and the sample fluid may act upon the whiteblood cells so as to align the intracellular features. In some cases,shear forces associated with velocity differentials between the sheathfluid and sample fluid may contribute to such alignment. These alignmenteffects may be impacted by a size differential between the particles andthe sample fluid ribbon as well. For example, where portions of theparticles extend out of the sample fluid ribbon and into the surroundingsheath fluid, shear forces associated with the difference in viscositymay have a pronounced effect on the intracellular feature alignment.

As depicted in FIG. 4L, portions of a cell such as a white blood cellcan extend into the sheath fluid. Embodiments of the present inventionencompass sheath fluid compositions that do not lyse or shred the cell,or otherwise compromise the integrity of the outer cell membrane, whenthe cell is exposed to the sheath fluid. A viscosity agent in the sheathfluid can operate to retain viability of cells in the sample fluidstream, so as to leave the structure (e.g. shape) and the content (e.g.nucleus) of the cells intact when the cell membrane or wall traverses aninterface between the sample fluid ribbon and the sheath fluid envelopeor otherwise extends from the sample fluid stream into the flowingsheath fluid.

Often, there are compressive forces acting upon the cells or particlesas they flow within the sample fluid ribbon along the flowcell. Hence,the cells may come into contact with the sheath fluid while the cellsare in a compressed state or are otherwise subject to compressive forcesas a result of a narrowing transition zone. The viscosity agent of thesheath fluid can operate to protect the compressed cells from beingshredded or destroyed when they emerge from the thin sample fluid ribbonand become exposed to the viscous sheath fluid, at least until the cellsreach the image capture site. Hence, the viscosity agent composition ofthe sheath fluid can operate as a cellular protectorant, while alsoenhancing alignment of the particles or intraparticle content.

With reference to FIGS. 4K and 4L, in some instances portions of thecell or particle may extend out of the thin sample fluid ribbon R andinto the surrounding sheath fluid. As discussed elsewhere herein, thesheath fluid may contain cellular protectants that inhibit or preventthe sheath fluid from disrupting or lysing the cells or particles. Forexample, the sheath fluid may contain cellular protectants that preservethe structural integrity of the cells walls as the cells are exposed tothe chemical environment of the sheath fluid. Similarly, the cellularprotectants may also operate to preserve the structural integrity of thecells walls as the cells experience any shear forces induced by flowcellgeometry, and a difference in velocity and/or viscosity between thesample fluid and the sheath fluid. Relatedly, the protectorants canprotect the cells or particles from forces resulting from the differencein velocity between the sample fluid and sheath fluid. In this way, thecells retain their viability as they reach the image capture site.

The shear forces can be significant at the interface between the samplefluid ribbon and the sheath fluid envelope. According to someembodiments, flow within the flowcell flowpath can be characterized by aparabolic flow profile. FIG. 4L-1 depicts exemplary aspects of parabolicflow profiles 4001-1 a and 4001-1 b. The parabolic profile 4001-1 a inthe upper panel is a typical velocity profile found in flows withincertain flowcell embodiments of the present invention (e.g. where thereis little or no viscosity differential between a sample fluid flowstreamthat is enveloped within a sheath fluid flowstream). As can be seen, ahighest linear velocity is observed in the middle of the fluid streamand slower linear velocities are observed near the flowcell wall.Profile 4001-1 a can also be observed in fluid stream with a slightviscosity difference between the sheath and sample fluids. In a casewhere there is a high viscosity differential between the sheath andfluid streams, a central bump is observed as shown in profile 4001-1 b,where there is a localized central area with amplified linearvelocities. According to some embodiments, particles that aresufficiently large in size will be subjected to some amount of shearforce, even where such particles are fully contained within a singlefluid phase (i.e. either within the sheath fluid envelope, oralternatively within the sample fluid ribbon).

In some instances, the velocity of the sheath fluid may be differentfrom the velocity of the sample fluid. For example, the sheath fluid maybe traveling at 80 mm/second and the sample fluid may be traveling at 60mm/second. Hence, in some instances, the sample fluid exits the distalcannula port at a sample fluid speed that is slower than the sheathfluid speed of the surrounding envelope. Hence, the sheath fluid canoperate to drag the sample fluid along the flowpath of the cannula, thusaccelerating the sample fluid and reducing the thickness of the samplefluid ribbon. The sample fluid ribbon maintains the overall volume andmass, so as it travels faster it becomes thinner. According to someembodiments, both the sheath fluid and the sample fluid have a velocityof between about 20 and 200 mm/second at the image capture site.

Typically, the velocity of the sample fluid increases as the samplefluid travels from the cannula exit port to the image capture site. Insome instances, the velocity of the sample fluid at the image capturesite is 40 times the velocity of the sample fluid as it exits thecannula port at the cannula distal portion. According to someembodiments, the decrease in cross sectional area of the sample ribbonis linear to the increase in velocity. According to some embodiments, ifthe sheath velocity at the cannula exit is higher than the sample ribbonvelocity this will also increase the final sample ribbon velocity at theimaging area.

The sheath fluid can operate to apply significant shear forces on thesample fluid ribbon and on particles within the sample fluid ribbon.Some forces are parallel to the direction of flow, and particles mayalso encounter forces which are perpendicular to the direction of flow.Often, as the sheath fluid and sample fluid approach the image capturesite or zone, the sheath and sample fluids are traveling at or near thesame velocity. Hence, the boundary or interface between the sheath andsample fluids as they pass the image capture site may present lowershear forces, as compared to the boundary or interface at the distalcannula exit port or at the tapered transition zone. For example, at thetapered transition zone, the boundary or interface between the sheathfluid envelope and sample fluid ribbon can be in transition, such thatthe sample ribbon which is initially slower and thicker becomes fasterand thinner, and particles in the sample fluid become more aligned. Putanother way, the shear forces may be prominent at the tapered transitionzone, and can dissipate toward the image capture site. The shear forcesat the image capture site can be represented by a parabolic profile, andcan be much lower than the shear forces at the tapered transition zone.Hence, cells or particles can experience higher shear forces as theypass through the transition zone, and lower shear forces as they passthrough the image capture site. According to some embodiments, theviscosity difference between the sheath and sample fluids can bring thered blood cells into alignment and thereby into focus. According to someembodiments, the viscosity difference between the sheath and samplefluids can bring white blood cell organelles into alignment and therebyinto focus. Relatedly, enhanced imaging results can be obtained forcellular and organelle components that are aligned and brought intofocus, resulting from the geometric narrowing of the stream and thevelocity difference between the sheath and sample fluids.

As noted elsewhere herein, and with reference to FIGS. 4K and 4L, as thesheath fluid and the sample fluid R flow through a reduction in flowpathsize or transition zone of a flowcell, and toward an imaging site 432 kor 432 l, a viscosity hydrofocusing effect induced by an interactionbetween the sheath fluid and the sample fluid R associated with aviscosity difference between the sheath fluid viscosity and the samplefluid viscosity, in combination with a geometric hydrofocusing effectinduced by an interaction between the sheath fluid and the sample fluidR associated with the reduction in flowpath size or transition zone,provides a target imaging state in at least some of the plurality ofparticles at the imaging site 432 k or 432 l.

In some cases, the target imaging state is a target orientation relativeto a focal plane F at the imaging site. For example, as depicted in FIG.4K-1, the particle (RBC) can be displaced at a distance from the focalplane F. In some cases, the target orientation involves a targetparticle orientation relative to the focal plane F at the imaging site432 k-1. The particle can be a blood cell, such as a red blood cell, awhite blood cell, or a platelet. As shown here, the flowpath at theimaging site 432 k-1 can define a P plane that is substantially parallelto or coplanar with the focal plane F. In some cases, a portion of theparticle may be positioned along the focal plane F, yet the centralportion of the particle may otherwise be offset from the focal plane F.In some cases, the target orientation involves a target positionrelative to the focal plane F at the imaging site 432 k-1. For example,the target position may involve positioning of the particle so that atleast a portion of the particle is disposed along the focal plane F. Insome cases, the target position may involve positioning of the particleso that a distance between the particle and the focal plane F does notexceed a certain threshold. In some cases, the target position involvesa target particle position that is relative to the focal plane F at theimaging site 432 k-1. In some cases, the target position is at or lessthan a distance D from the focal plane F, where distance D correspondsto a positional tolerance. A viscosity differential between the sheathfluid and the sample fluid can be selected so as to achieve a desiredpositioning of the ribbon sample stream within the flowcell (e.g.relative to flowpath plane P and/or focal plane F). In some cases, theviscosity differential can be selected so as to achieve a targetparticle position that is at or less than the positional tolerance D.

In some cases, the focal plane F has a thickness or depth of field asindicated in FIG. 4K-2, and the particle (RBC) has a target imagingstate relative to the focal plane thickness. For example, the targetposition for the particle can be within the focal plane F or at leastpartially within the focal plane F. In some cases a high opticalresolution imaging device or camera can have a depth of field or focalplane thickness of about 7 μm. In some cases, the depth of field orfocal plane thickness has a value with a range from about 2 μm to about10 μm. In some cases, the depth of the field of the camera is similar orequal to the sample ribbon thickness at the image capture site.

In some cases, the target orientation can involve a target alignmentrelative to the focal plane F at the imaging site. For example, thetarget alignment can indicate that a plane defined by the particle isaligned with the focal plane F, not to exceed a certain angle α relativeto the focal plane F at the image capture site 432 k-3 as shown in FIG.4K-3. In some cases, the target imaging state can involve a limitationon the number or percentage of misaligned particles in a sample. Forexample, a difference in viscosity between the sheath fluid and thesample fluid R can be effective to limit red blood cells imagingorientation misalignment in the blood fluid sample to less than about10%. That is, 90 or more red blood cells out of 100 red blood cells in asample can be aligned so that their major surfaces face toward theimaging device (as depicted in FIGS. 4K-1 and 4K-2) or so that thealignment of those 90 or more RBCs is within 20 degrees from a planesubstantially parallel to the direction of flow (e.g. RBC alignmentangle α is 20 degrees or less). As discussed elsewhere herein, in somecases at least 92% of non-spherical particles such as RBCs can bealigned in a plane substantially parallel to the direction of flow. Insome cases, at least between 75% and 95% of non-spherical particles suchas RBCs can be substantially aligned, namely within 20 degrees from aplane substantially parallel to the direction of flow (e.g. alignmentangle α is 20 degrees or less). According to some embodiments, 90% ormore of certain particles (e.g. red blood cells and/or platelets) can beoriented transverse to the imaging axis of the imaging device.

In some cases, embodiments of the present invention include compositionsfor use with a hematology system as described herein, such as a sheathfluid or particle and intracellular organelle alignment liquid (PIOAL).Such sheath fluids or PIOALs are suitable for use in a combinedviscosity and geometric hydrofocusing visual analyzer. The PIOAL canoperate to direct or facilitate flow of a blood sample fluid of a givenviscosity through a narrowing flowcell transition zone of the visualanalyzer. The PIOAL can include a fluid having a higher viscosity thanthe viscosity of the sample. A viscosity hydrofocusing effect induced byan interaction between the PIOAL fluid and the sample fluid associatedwith the viscosity difference, in combination with a geometrichydrofocusing effect induced by an interaction between the PIOAL fluidand the sample fluid associated with the narrowing flowcell transitionzone, can be effective to provide a target imaging state in at leastsome of the plurality of particles at an imaging site of the visualanalyzer while retaining viability of cells in the blood sample fluid.

FIG. 4M depicts an exemplary neutrophil 400 m (a type of white bloodcell) having internal organelles such as lobes 410 m. As a result of theviscosity differential between the sample fluid and the sheath fluid,the internal organelles can align within the cell, as indicated by FIG.4N. Hence, the intracellular organelles can be effectively imaged withan image capture device 430 m, without the organelles overlapping oneanother. That is, instead of the lobes being stacked upon one another asdepicted in FIG. 4M, when viewed from the imaging or optical axis of theimage capture device the lobes are aligned and sitting side by side asdepicted in FIG. 4N. Hence, the lobes can be visualized in the capturedimaged more effectively. The internal organelle alignment is asurprising and unexpected result of the viscosity differential betweenthe sample and sheath fluids. Accordingly, enhanced imaging resultscorresponding to cell alignment and in-focus are achieved using theviscosity differential, hydrodynamic flow, and geometric compressionfeatures.

As noted elsewhere herein, and with reference to FIGS. 4M and 4N, as thesheath fluid and the sample fluid R flow through a reduction in flowpathsize or transition zone of a flowcell, and toward an imaging site of animage capture device 430 m or 430 n, a viscosity hydrofocusing effectinduced by an interaction between the sheath fluid and the sample fluidR associated with a viscosity difference between the sheath fluidviscosity and the sample fluid viscosity, in combination with ageometric hydrofocusing effect induced by an interaction between thesheath fluid and the sample fluid R associated with the reduction inflowpath size or transition zone, provides a target imaging state in atleast some of the plurality of particles at the imaging site. Accordingto some embodiments, the target imaging state may correspond to adistribution of imaging states.

In some cases, the target imaging state can involve a targetintraparticle structure orientation (e.g. alignment and/or position)relative to a focal plane at the imaging site. For example, as depictedin FIG. 4N, the internal structures 410 m (e.g. intracellular structure,organelle, lobe, or the like) can be oriented relative to the focalplane F. In some cases, the target alignment involves a targetintraparticle structure alignment relative to a focal plane F at theimaging site, similar to the particle alignment relationship depicted inFIG. 4K-3. In some cases, the target position involves a targetintraparticle structure position relative to a focal plane at theimaging site, similar to the particle position relationship depicted inFIG. 4K-1. In some cases, the target orientation of the intraparticlestructure can include both a target alignment relative to the focalplane and also a target position relative to the focal plane. In somecases, the target imaging state can involve a target deformation at theimaging site. For example, as depicted in FIG. 4N, the particle 400 mhas a compressed shape as compared to the particle shape depicted inFIG. 4M. Hence, it can be seen that operation of the flowcell canproduce a lateral compression effect on the particle shapes. Relatedly,the intraparticle features can be positionally or directionally oriented(e.g. aligned with respect to the focal plane F and/or ribbon flowplane) as the particle itself is compressed in shape. According to someembodiments, a velocity difference between the sheath and sample fluidscan produce friction within the flowstream, and a viscosity differencebetween the sheath and sample fluids can amplify that hydrodynamicfriction.

Examples

Any of a variety of hematology or blood particle analysis techniques canbe performed using images of sample fluid flowing through the flowcell.Often, image analysis can involve determining certain cell or particleparameters, or measuring, detecting, or evaluating certain cell orparticle features. For example, image analysis can involve evaluatingcell or particle size, cell nucleus features, cell cytoplasm features,intracellular organelle features, and the like. Relatedly, analysistechniques can encompass certain counting or classification methods ordiagnostic tests, including white blood cell (WBC) differentials. Insome cases, images obtained using the flowcell can support a 5-part WBCdifferential test. In some cases, images obtained using the flowcell cansupport a 9-part WBC differential test. Relatedly, with reference toFIG. 4, the processor 440 can include or be in operative associationwith a storage medium having a computer application that, when executedby the processor, is configured to cause the system 400 to differentiatedifferent types of cells based on images obtained from the image capturedevice. For example, diagnostic or testing techniques can be used todifferentiate various cells (e.g. neutrophils, lymphocytes, monocytes,eosinophils, basophils, metamyelocytes, myelocytes, promyelocytes, andblasts).

The Examples provided herein are for the purpose of illustration only,and the invention is not limited to these Examples, but ratherencompasses all variations that are evident as a result of the teachingprovided herein.

Prior to the experiments described herein, there was no publishedprotocol that allows for the development and the methods of usecomprising PIOAL for aligning particles and repositioning intracellularcontent as disclosed herein. This is useful for image-based analysis anddifferential categorization and subcategorization of particles in bodyfluid (e.g. blood) samples. The methods and compositions disclosedherein can optionally stain and/or lyse particles in a suitable mannerto achieve white cell staining, reticulocyte staining and plateletstaining, that mimic's Wright stained cells seen on a whole blood smear.

The exemplary compositions described herein allow staining to occurs ata relatively low blood to reagent dilution and the staining can occursrapidly (e.g. within 30 sec). If desired, the exemplary method canemploy the use of a surfactant in combination with heat to achieve redcell lysis. The exemplary formulations can be modified to retain RBCintegrity and still achieve WBC, retic and platelet staining efficacy.

Aspects and embodiments of the present disclosure are based on thesurprising and unexpected discovery that certain PIOAL compositions haveunexpected properties aligning cells and re-positioning intracellularstructures when used to perform image-based particle/cell analysis.

By way of example, several exemplary PIOAL formulations and methods ofuse thereof were developed. The following are some exemplars of PIOALformulations with the desired properties.

FIG. 4O shows a comparison of images obtained using PIOAL versus imagesobtained using a non PIOAL sheath fluid. Use of the PIOAL resulted inmore in-focus cellular contents such as lobes, cytoplasm, and/orgranule. In this example, a PIOAL comprising a viscosity agent (about30% glycerol) was used to process the sample. The pH was adjusted to apH of about 6.8 to 7.2 and the sample mixture was made isotonic by (0.9%sodium chloride). The results shown here demonstrate the efficacy of anexemplary PIOAL used on an image analyzer to align cells andintracellular organelles.

FIGS. 4P and 4Q show a comparison of images obtained using a standardsheath fluid (FIG. P upper and lower panels) versus images obtainedusing an exemplary PIOAL fluid (FIG. 4Q upper and lower panels). Asshown here, the use of PIOAL resulted in an improved RBC alignment, forexample by orienting the major surfaces of the red blood cells to facetoward the camera or imaging device. The sample was analyzed using aninstrument focusing protocol (on an exemplary target 44 as depicted inFIG. 1) and the target was brought into focus by a visual analyzer. Thefocusing system was then offset by displacement distance 52, resultingin the particles in the ribbon-shaped sample stream being in focus. Theblood sample was previously diluted using a sample diluent. The sampleflowed through a cannula and along a flowpath of a flowcell, therebygenerating a ribbon-shaped sample stream (e.g. 2 microns in thickness)which was between two layers of PIOAL or standard sheath (in controls).The visual analyzer then generates focused images of the particles inthe ribbon-shaped sample stream (e.g. at about 60 frames per second) tobe used for analysis. The blood sample is obtained from a subject andprocessed for analysis by the blood analyzer. Images of RBCs in aflowcell are captured while the sample is processed using a standardsheath fluid or a PIOAL. Relative percentages demonstrate significantimprovement in the number of aligned RBCs based on imaging data (e.g. 4Pand 4Q). The result demonstrated that PIOAL was efficacious atincreasing the percentage of RBC alignment while in flow in theribbon-shaped sample stream using the focusing instrument/protocols asdescribed herein.

It was also observed that the implementation of PIOAL results inimproved alignment based on using increasing levels of glycerol (gly) insymmetric and asymmetric flowcells.

The chart in FIG. 4R shows the percentage of non-aligned cells obtainedusing 0%-30% glycerol in the PIOAL with symmetric vs. asymmetric flowcells. Using 30% glycerol in the PIOAL and a symmetric flowcell resultsin reducing the percentage of misaligned cells to only 8%. Note withoutglycerol in the PIOAL, and with an asymmetric cell, the percentage ofmisaligned cells increased to 87%. Hence, this chart demonstrates theeffect of glycerol percentage and flowcell geometry on particle (e.g.RBC) alignment. The addition of glycerol decreases the percentage ofmisaligned RBC cells using either symmetric or asymmetric flowcellgeometry. The % non-aligned RBCs was reduced from 87% down to 15% in theasymmetric and 46% to 8% in symmetrical cells. Thus, the chart providesa comparison between misalignment results (8%) obtained from an analyzerconfiguration that involves a symmetrical narrowing flowcell transitionzone and a viscous sheath fluid and misalignment results (46%) obtainedfrom an analyzer configuration that involves a symmetrical narrowingflowcell transition zone without the use of a viscous sheath fluid.

These results provide evidence for the surprising and unexpecteddiscovery that certain PIOAL compositions have unexpected propertiesaligning cells and re-positioning intracellular structures when used toperform image-based particle/cell analysis.

By way of example, several exemplary PIOAL formulations and methods ofuse thereof were developed. The following are some exemplars of PIOALformulations with the desired properties. The PIOAL comprises a diluentand at least one viscosity modifying agent.

Exemplary PIOAL formulation A includes a 30% (v/v) glycerol solutionhaving 300 mL glycerol and QS (quantity sufficient or to bring the finalvolume up to) to 1 L with diluent containing 9.84 g sodium sulfate, 4.07g sodium chloride, 0.11 g Procaine HCl, 0.68 g potassium phosphatemonobasic, 0.71 g sodium phosphate dibasic, and 1.86 g disodium EDTA.The initial mixture was followed by QS to 1 L with deionized water whileadjusting pH to 7.2 with sodium hydroxide.

Exemplary PIOAL formulation B includes a 6.5% (v/v) glycerol solutionhaving 65 mL glycerol and QS to 1 L with suitable exemplary diluentcontaining 9.84 g sodium sulfate, 4.07 g sodium chloride, 0.11 gProcaine HCl, 0.68 g potassium phosphate monobasic, 0.71 g sodiumphosphate dibasic, and 1.86 g disodium EDTA. The initial mixture wasfollowed by QS to 1 L with deionized water while adjusting pH to 7.2with sodium hydroxide.

Exemplary PIOAL formulation C includes a 5% glycerol (v/v) solution with1% PVP (w/v) in buffer having 50 mL glycerol, 10 g PVP (MW: 360,000), 1packet of Sigma PBS powder, at pH 7.4 (0.01M phosphate buffered saline;0.138M sodium chloride; 0.0027M potassium chloride), and QS to 1 L withdeionized water.

Exemplary PIOAL formulation D includes a 1.6% PVP (w/v) solution having16 g PVP (MW: 360,000) and 1 packet of Sigma PBS powder, at pH 7.4(0.01M phosphate buffered saline; 0.138M sodium chloride; 0.0027Mpotassium chloride), and QS to 1 L with deionized water.

FIGS. 5A and 5B depict exemplary flowstream characteristics related toshear force, lateral compression, orientation, differential viscosity,relative movement between sheath and sample fluids, and the like.

Methods

FIG. 6 depicts aspects of an exemplary method 600 for imaging aplurality of particles using a particle analysis system configured forcombined viscosity and geometric hydrofocusing according to embodimentsof the present invention. The particles can be included in a blood fluidsample 610 having a sample fluid viscosity. As shown here, the methodcan include flowing a sheath fluid 620 along a flowpath of a flowcell asindicated by step 630. The sheath fluid 620 can have a sheath fluidviscosity that differs from the sample fluid viscosity by a viscositydifference in a predetermined viscosity difference range. The method canalso include injecting the blood fluid sample 610 into the flowingsheath fluid within the flowcell, as indicated by step 630, so as toprovide a sample fluid stream enveloped by the sheath fluid. Further,the methods can include flowing the sample fluid stream and the sheathfluid through a reduction in flowpath size toward an imaging site asindicated by step 640. As the sample stream and sheath fluids passthrough the reduction in flowpath size or narrowing transition zone, aviscosity hydrofocusing effect induced by an interaction between thesheath fluid and the sample fluid stream associated with the viscositydifference (as depicted in step 650), in combination with a geometrichydrofocusing effect induced by an interaction between the sheath fluidand the sample fluid stream associated with the reduction in flowpathsize (as depicted in step 660), is effective to provide a target imagingstate in at least some of the plurality of particles at the imaging sitewhile a viscosity agent in the sheath fluid retains viability of cellsin the sample fluid stream leaving structure and content of the cellsintact when the cells extend from the sample fluid stream into theflowing sheath fluid as depicted by step 670. Methods may also includeimaging the plurality of particles at the imaging site, as depicted bystep 680.

Shear Strain Rate

FIGS. 7 and 8 depict aspects of shear strain rate values for certainflow conditions in a flowcell according to embodiments of the presentinvention. In each of these drawings, a 30% glycerol sheath fluid isused. In some cases, the viscosity can have a value of 2.45×10⁻³. Ashear stress value can be equal to the product obtained by multiplying aviscosity value with a strain rate value. With regard to FIG. 7, thesample can have a flow rate of 0.3 μL/sec and the sheath fluid can havea flow rate of 21 μL/sec. With regard to FIG. 8, the sample can have aflow rate of 1 μL/sec and the sheath fluid can have a flow rate of 70μL/sec. In each of these figures, it can be seen that the flow presentsa lower strain value toward the center (C) and a higher strain valuetoward the periphery (P). Such strain values can correspond to anasymmetric flowcell configuration, in some embodiments.

As depicted in FIG. 7, according to some embodiments, the lower strainrate toward the center (C) portion of the flowstream can have a value ofabout 500 (1/s) or lower and the higher strain rate toward the periphery(P) of the flowstream can have a value of about 3000 (1/s) or higher. Asdepicted in FIG. 8, according to some embodiments, the lower strain ratetoward the center (C) portion of the flowstream can have a value ofabout 1000 (1/s) or lower and the higher strain rate toward theperiphery (P) of the flowstream can have a value of about 9000 (1/s) orhigher.

Hence, it can be seen that lower sample and sheath fluid rates (e.g.FIG. 7) correspond to lower strain rates, and higher sample and sheathfluid rates (e.g. FIG. 8) correspond to higher strain rates. It isunderstood that embodiments of the present invention encompass the useof sample and/or sheath fluids corresponding to various viscosityvalues, various strain rate values, and/or various shear stress values.

According to some embodiments, the PIOAL has a suitable viscosity anddensity, and flow rates at the point of introduction to the flowcell ofthe sample are such that the sample fluid flattens into a thin ribbon.The ribbon-shaped sample stream is carried along with the PIOAL, to passin front of a viewing port where an objective lens and a light sourceare arranged to permit viewing of the ribbon-shaped sample stream. Thesample fluid is introduced, for example, injected at a point where theflowpath of the PIOAL narrows symmetrically. As a result, the samplefluid stream is flattened and stretched into a thin ribbon. A PIOAL ofthis disclosure may be used as the sheath fluid with any visual analyzerof this disclosure. In one embodiment, the PIOAL can be introduced intoan end of the flowcell to carry along the sample fluid toward thedischarge.

The dimension of the ribbon-shaped sample stream in the viewing zone isaffected by geometric thinning of the PIOAL flowpath and differentiallinear velocity of the sample fluid and PIOAL resulting in thinning andstretching of the ribbon-shaped sample stream. The initial differentiallinear velocity of the sample to PIOAL may range from 0.5:1 to 5:1. ThePIOAL flowpath cross section may be thinned by reducing the depth by afactor of about 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1,55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, 100:1, 105:1,110:1, 115:1, 125:1, 130:1, 140:1, 150:1, 160:1, 170:1, 180:1, 190:1, or200:1. In one embodiment, the geometric thinning is 40:1. In oneembodiment, the geometric thinning is 30:1. Factors taken into accountare transit time through the flowcell, desired rate of samplethroughput, achieving a ribbon-shaped sample stream thickness comparableto particle size, obtaining alignment of particles and organelles,achieving in focus content of particles, balancing pressure, flow, andviscosity within operational limits, optimizing ribbon-shaped samplestream thickness, obtaining a desired linear velocity, manufacturabilityconsiderations, and volumes of sample and PIOAL required.

The length and volume of the cannula and the cross-section flatteningmay be selected to reduce the period of sample flow instability, therebyincreasing throughput. In some embodiments the period of flowinstability may be less than about 3, 2.75, 2.5, 2.25, 2, 1.75, 1.51.25, or less than about 1 second. A smaller cannula volume may alsoreduce the time and volume of diluent needed to clean the cannulabetween sample runs. In some embodiments the transit time through theflowcell is 1, 2, 3, or 4 seconds, or any range in between any two ofthose times. In some embodiments the transit time may be less than 4, 3or 2 seconds.

The viscosities and the flow rates of the sample fluid and the PIOAL andthe contour of the flowcell are arranged such that the PIOAL flowflattens and stretches the sample flow into a flat ribbon consistentlythrough the viewing zone at a dependable location corresponding to animage capture site. The sample fluid stream may be compressed toapproximately 2 to 3 μm in fluid flow thickness. Several blood celltypes have diameters larger than the stream thickness. Sheer forces inthe direction parallel to the direction of the flow cause an increase ofan image projection of the particles under imaging conditions in thefocal plane of the high optical resolution imaging device and/or causingthe intraparticle structures, for example, intracellular structures,organelles or lobes, to be positioned, repositioned, and/orbetter-positioned to be substantially parallel to the direction of flow.The high optical resolution imaging device depth of field is up to 7 μm,for example, 1-4 μm.

The flow cross section of the PIOAL, with the ribbon-shaped samplestream carried along, is constant through a viewing zone in front of aviewing port through which the objective lens is directed. The objectivelens may be the objective component of a high optical resolution imagingdevice or the digital image capture device. The ribbon-shaped samplestream follows a path across the viewing zone at a known and repeatableposition within the flowcell, for example, at a known and repeatabledistance from two walls of the flowcell, being discharged downstream.

Optical information from the particles in the sample are detected by adetecting section in the analyzer, when the ribbon-shaped sample streamis carried through the viewing zone in front of the viewing port,thereby generating data from the particles/cells contained in thesample. The use of this analyzer allows capture, processing,categorization and subcategorization and counting of cells and/orparticles contained in samples. The PIOAL liquid can be prepared by theaddition of viscosity modifying agent, buffer agent, pH adjusting agent,antimicrobial agent, ionic strength modifier, surfactant, and/or achelating agent. Exemplary functional components and/or features of theanalyzer in the present disclosure can include, for example, the abilityto acquire and/or process data from image analysis, sample stainingprocessing, image processing, and/or particle image identification,counting, and/or categorization and subcategorization.

In one embodiment this disclosure was based on the surprising andunexpected discovery that the addition of a suitable amount of aviscosity agent in the PIOAL significantly improves particle/cellalignment in a flowcell, leading to a higher percentage of alignedcells, or cellular components in focus, and higher quality images ofcells and/or particles in flow. A viscosity differential in combinationwith a geometric focusing effect of a narrowing transition zone canachieve enhanced alignment and focus results. Improved results can beseen with a velocity differential between the sheath and sample fluidstreams. In some cases, improved images with no overlaps of cells andparticles are observed when the sample fluid is delivered at a certainrate. The addition of the viscosity agent increases the shear forces oncells like RBCs, which improves the alignment of the cells in a planesubstantially parallel to the flow direction, which results in imageoptimization. This also results in positioning, repositioning, and/orbetter-positioning of intraparticle structures such as intracellularstructures, organelles or lobes substantially parallel to the directionof flow, which results in image optimization. The viscosity agent alsoreduces misalignment of cells, generally, but not limited to cells thatare smaller in diameter than the flow stream.

Alignment of cells that are smaller in diameter than the flow stream,for example, red blood cells may be obtained by for example, increasingthe viscosity of the PIOAL, or by increasing the flow speed ratio. Thisresults in alignment of the RBCs parallel to the direction of the flowand to the focal plane FP (e.g. as depicted in FIG. 4K). In someembodiments, a reduction in RBC misalignment and/or increase in RBCalignment is achieved by increasing the viscosity of the PIOAL.

The ribbon-shaped sample stream thickness can be affected by therelative viscosities and flow rates of the sample fluid and the PIOAL.The source of the sample and/or the source of the PIOAL, for examplecomprising precision displacement pumps, can be configured to providethe sample and/or the PIOAL at controllable flow rates for optimizingthe dimensions of the ribbon-shaped sample stream, namely as a thinribbon at least as wide as the field of view of the high opticalresolution imaging device or the digital image capture device.

The flow cross section of the PIOAL, with the ribbon-shaped samplestream carried along, is constant through a viewing zone in front of aviewing port through which the high optical resolution imaging device isdirected. The ribbon-shaped sample stream follows a path across theviewing zone at a known and repeatable distance from either of the frontand rear walls of the flowcell, being discharged downstream of that.

The present disclosure provides a technique for automatically achievinga correct working position of the high optical resolution imaging devicefor focusing on the ribbon-shaped sample stream. The flowcell structureis configured such that the ribbon-shaped sample stream has a fixed andrepeatable location between the walls of the flowcell defining the flowpath of sample fluid, in a thin ribbon between layers of PIOAL, passingthrough a viewing zone in the flowcell. In the flowcell embodimentsdisclosed, for example in FIG. 1-4G, the cross section of the flowpathfor the PIOAL can narrow symmetrically at a transition zone, and asample can be inserted through a flattened orifice such as a tube with arectangular lumen at the orifice. The narrowing flowpath (for examplegeometrically narrowing in cross sectional area by a ratio of 20:1 to40:1) and also due to an optionally greater linear velocity of the PIOALcompared to the flow of the sample, cooperate to flatten the samplecross section by a ratio of about 20:1 to 70:1. According to someembodiments, the ratio can be within a range from 10:1 to 100:1, withina range from 50:1 to 100:1, within a range from 70:1 to 80:1. Accordingto some embodiments, the ratio is 75:1. Effectively, due to thecombination of flow rate, viscosity, and geometry, the sample is formedinto a thin ribbon. The narrowing flowpath (for example geometricallynarrowing in cross sectional area by a ratio of 40:1, or by a ratiobetween 20:1 to 70:1) and a difference in linear speed of the PIOALcompared to the flow of the sample, cooperate to compress the samplecross section by a ratio of about 20:1 to 70:1. In some embodiments thecross section thickness ratio may be 40:1. In some embodiments the crosssection thickness ratio may be 30:1.

As a result, process variations such as the specific linear velocitiesof the sample and the PIOAL, do not tend to displace the ribbon-shapedsample stream from its location in the flow. Relative to the structureof the flowcell, the ribbon-shaped sample stream location is stable andrepeatable.

In another aspect, this invention relates to a kit comprising theparticle contrast agent compositions of this invention. The kit may alsocontain instructions on the use of particle contrast agent compositionaccording to any of the methods described herein. The kit may alsoinclude a particle and/or intracellular organelle alignment liquid(PIOAL). The kit may also contain a programmable storage medium andrelated software for image based identification of particles such asneutrophil, lymphocytes, monocyte, eosinophils, basophils, platelets,reticulocytes, nucleated RBCs, blasts, promyelocytes, myelocytes,metamyelocytes, bacteria, fungi, protists, protozoa, or parasites. Thekit may also comprise one or more buffers, which may include isotonicbuffers and/or diluents. The kit and or buffer may further comprise asurfactant, a pH adjusting agent, and/or an antimicrobial agent. Inother embodiments, the kit may also comprise a cleaning or flushingsolution. The kit may also comprise standards for positive and negativecontrols. In some embodiments the standard may comprise a standardstained cell reagent. The kit may also comprise disposables such asdisposable micropipettes, tips or tubes for transferring the componentsof the kit. The kit may contain any one, or any combination of two ormore of these kit components.

The discrimination of blood cells in a blood sample is an exemplaryapplication for which embodiments of the instant invention areparticularly well suited. The sample is prepared by automated techniquesand presented to a high optical resolution imaging device as a thinribbon-shaped sample stream to be imaged periodically while theribbon-shaped sample stream flows across a field of view. The images ofthe particles (such as blood cells) can be distinguished from oneanother, categorized, subcategorized, and counted, using pixel imagedata programmed processing techniques, either exclusively automaticallyor with limited human assistance, to identify and count cells orparticles. In addition to the cell images, which can be stored and madeavailable in the case of unusual or critical features of particles, theoutput data includes a count of the occurrences of each particularcategory and/or subcategory of cell or particle distinguished in therecorded sample images.

The counts of the different particles found in each image can beprocessed further, for example used to accumulate accurate andstatistically significant ratios of cells of each distinguished categoryand/or subcategory in the sample as a whole. The sample used for visualdiscrimination can be diluted, but the proportions of cells in eachcategory and/or subcategory are represented in the diluted sample,particularly after a number of images have been processed.

The apparatus, compositions, and methods disclosed herein are useful indiscriminating and quantifying cells in samples based on visualdistinctions. The sample can be a biological sample, for example, a bodyfluid sample comprising white blood cells, including without limitation,blood, serum, bone marrow, lavage fluid, effusions, exudates,cerebrospinal fluid, pleural fluid, peritoneal fluid, and amnioticfluid. In some embodiments, the sample can be a solid tissue sample,e.g., a biopsy sample that has been treated to produce a cellsuspension. The sample may also be a suspension obtained from treating afecal sample. A sample may also be a laboratory or production linesample comprising particles, such as a cell culture sample. The termsample may be used to refer to a sample obtained from a patient orlaboratory or any fraction, portion or aliquot thereof. The sample canbe diluted, divided into portions, or stained in some processes.

In one aspect, the systems, compositions and methods of this disclosureprovide surprisingly high quality images of cells in a flow. In oneaspect, the visual analyzer can be used in methods of this disclosure toprovide automated image based WBC differential counting. In certainembodiments, the methods of this disclosure relate to automatedidentification of visual distinctions, including morphological featuresand/or abnormalities for determining, diagnosing, prognosing,predicting, and/or supporting a diagnosis of whether a subject ishealthy or has a disease, condition, abnormality and/or infection and/oris responsive or non-responsive to treatment. The system may furthercomprise a particle counter in some embodiments. Applications includecategorizing and/or subcategorizing, and counting cells in a fluidsample, such as a blood sample. Other similar uses for countingadditional types of particles and/or particles in other fluid samplesare also contemplated. The system, compositions, and methods of thisinvention can be used for real-time categorization and subcategorizationand viewing of images using any suitable automated particle recognitionalgorithm. The captured images for each sample can be stored to beviewed at a later date.

In another aspect, the apparatus, compositions, and methods of thisinvention provide surprisingly more accurate image based cellcategorization and subcategorization and flagging which reduces themanual review rate compared to the manual review rate when using currentautomated analyzers. The systems, compositions, and methods reduce themanual review rate and permit the manual review to be performed on theinstrument. In addition, the systems, compositions, and methods of thisdisclosure also reduce the percentage of samples flagged duringautomated analysis as requiring manual review.

The present disclosure further relates to systems, methods andcompositions for combining a complete blood count (CBC) counter with ananalyzer, such as a visual analyzer, in order to obtain a CBC and animage based expanded white blood cell differential count and an imagebased expanded platelet count, thereby extending the effective detectionrange for counting platelets.

Accordingly, in some embodiments, the present disclosure provides anapparatus and a method for analyzing a sample containing particles, forexample, blood cells. According to this disclosure, a visual analyzer isprovided for obtaining images of a sample comprising particles suspendedin a liquid. In some embodiments, the visual analyzer comprises aflowcell and an autofocus component, in which a liquid sample containingparticles of interest is caused to flow through a flowcell having aviewport through which a camera coupled to an objective lens capturesdigital images of particles. Exemplary autofocus techniques which can beimplemented using embodiments of the present invention are disclosed inco-pending U.S. patent application Ser. No. 14/216,811, the content ofwhich is incorporated herein by reference. The flowcell is coupled to asource of sample fluid, such as a diluted and/or treated blood sample orother bodily fluid sample as described herein, and to a source of aclear sheath fluid, or particle and/or intracellular organelle alignmentliquid (PIOAL).

In one embodiment, the apparatus also comprises a particle counterhaving at least one detection range, as well as an analyzer, and aprocessor. The analyzer and the processor are configured to provideadditional information to correct counting, categorization, andsubcategorization errors associated with the particle counter, andfurther determine accurate particle count or concentration of differentcategories and/or subcategories of particles in the sample.

The instant disclosure provides methods and compositions useful forparticle and/or intracellular organelle alignment in conductingimage-based sample analysis. In some embodiments, this disclosurerelates to methods and compositions for combined counting and imagingsystem with the ability to perform a complete blood count (CBC) and animage based expanded white blood cell (WBC) differential able toidentify and count cell types, such as WBCs, RBCs, and/or platelets,including, for example, neutrophils, lymphocytes, monocytes,eosinophils, basophils, reticulocytes, nucleated RBCs, blasts,promyelocytes, myelocytes, or metamyelocytes, and to provide image basedinformation for WBC counts and morphologies, red blood cell (RBC) countsand morphologies and platelet (PLT) counts and morphologies.

In other embodiments, this disclosure relates to a PIOAL that can beused in image based analysis of particles as described herein. Cellcategory and/or subcategory count in blood samples is used in thisdisclosure as nonlimiting examples of the sort of samples that may beanalyzed. In some embodiments, cells present in samples may also includebacterial or fungal cells as well as white blood cells, red blood cellsand/or platelets. In some embodiments, particle suspensions obtainedfrom tissues or aspirates may be analyzed.

In some aspects, samples are presented, imaged and analyzed in anautomated manner. In the case of blood samples, the sample may besubstantially diluted with a suitable diluent or saline solution, whichreduces the extent to which the view of some cells might be hidden byother cells in an undiluted or less-diluted sample. The cells can betreated with agents that enhance the contrast of some cell aspects, forexample using permeabilizing agents to render cell membranes permeable,and histological stains to adhere in and to reveal features, such asgranules and the nucleus. In some embodiments it may be desirable tostain an aliquot of the sample for counting and characterizing particleswhich include reticulocytes, nucleated red blood cells, and platelets,and for white blood cell differential, characterization and analysis. Inother embodiments, samples containing red blood cells may be dilutedbefore introduction to the flowcell and imaging.

According to some embodiments, the particulars of sample preparationapparatus and methods for sample dilution, permeabilizing andhistological staining, generally are accomplished using precision pumpsand valves operated by one or more programmable controllers, and are notcentral to this disclosure. Examples can be found in patents assigned toInternational Remote Imaging Systems, Inc., such as U.S. Pat. No.7,319,907, concerning programmable controls. Likewise, techniques fordistinguishing among certain cell categories and/or subcategories bytheir attributes such as relative size and color can be found in U.S.Pat. No. 5,436,978 in connection with white blood cells. The disclosuresof these patents are hereby incorporated by reference. According to someembodiments, sample preparation techniques may include staining, lysing,permeabilizing, and other processing modalities such as those describedin co-pending U.S. patent application Ser. No. 14/216,339, the contentof which is incorporated herein by reference.

The term high optical resolution imaging device can include devices thatare capable of obtaining particles images with sufficient visualdistinctions to differentiate morphological features and/or changes.Exemplary high optical resolution imaging devices can include deviceswith an optical resolution of 1 μm or lower, including for example, 0.4to 0.5 μm, such as for example, 0.46 μm.

In some embodiments, the images obtained in any of the compositionsand/or methods of this invention may be digitized images. In someembodiments, the images obtained are microscopy images. In certainembodiments, the images may be obtained manually. In other embodiments,at least part of the procedure for obtaining the images is automated. Insome embodiments, the images may be obtained using a visual analyzercomprising a flowcell, a high optical resolution imaging device or thedigital image capture device, optionally with an autofocus feature.

In one embodiment, the images provide information relating to thecytosolic, cell nucleus and/or nuclear components of the cell. In oneembodiment, the images provide information relating to the granularcomponent and/or other morphological features of the cell. In oneembodiment, the images provide information relating to cytosolic,nuclear and/or granular components of the cell. The granular and/ornuclear images and/or features are determinative for cell categorizationand subcategorization both independently or in combination with eachother.

In yet another aspect, the methods of this invention relate to a methodfor performing image-based red blood cell categorization andsubcategorization comprising: a) imaging a portion of the red bloodcells; and b) determining the morphology of the imaged red blood cells.As used herein, red blood cells (RBC) can include, for example, normalor abnormal red blood cells, reticulocytes, nucleated red blood cells,and/or malaria-infected cells. In some embodiments, the imaging isperformed using the apparatus of this disclosure such as an apparatuscomprising a particle counter, a visual analyzer and a processor.

As used herein, an exemplary complete blood count (CBC) can include atest panel typically requested by a doctor or other medical professionalthat provides information about the particles and/or cells in apatient's blood sample. Exemplary cells that circulate in thebloodstream can be generally divided into three types: including but notlimited to, for example, white blood cells (e.g., leukocytes), red bloodcells (e.g., erythrocytes), and platelets (e.g., thrombocytes).

As used herein, abnormally high or low counts may indicate the presenceof disease, disorder, and/or condition. Thus, a CBC is one of thecommonly performed blood tests in medicine, as it can provide anoverview of a patient's general health status. Accordingly, a CBC isroutinely performed during annual physical examinations.

As used herein, typically a phlebotomist collects the blood sample fromthe subject, the blood is generally drawn into a test tube typicallycontaining an anticoagulant (e.g., EDTA, sometimes citrate) to stop itfrom clotting. The sample is then transported to a laboratory. Sometimesthe sample is drawn off a finger prick using a Pasteur pipette forimmediate processing by an automated counter. In one embodiment, theparticle image is acquired while the particle is enveloped in a sheathfluid or PIOAL. In certain embodiments, the blood sample may be viewedon a slide prepared with a sample of the patient's blood under amicroscope (a blood film, or peripheral smear). In certain embodiments,the complete blood count is performed by an automated analyzer.

As used herein, data/parameters of a blood count can include, forexample, total red blood cells; hemoglobin—the amount of hemoglobin inthe blood; hematocrit or packed cell volume (PCV); mean corpuscularvolume (MCV)—the average volume of the red cells (anemia is classifiedas microcytic or macrocytic based on whether this value is above orbelow the expected normal range. Other conditions that can affect MCVinclude thalassemia, reticulocytosis and alcoholism); mean corpuscularhemoglobin (MCH)—the average amount of hemoglobin per red blood cell, inpicograms; mean corpuscular hemoglobin concentration (MCHC)—the averageconcentration of hemoglobin in the cells; red blood cell distributionwidth (RDW)—the variation in cellular volume of the RBC population;total white blood cells; neutrophil granulocytes (may indicate bacterialinfection, typically increased in acute viral infections). Due to thesegmented appearance of the nucleus, neutrophils are sometimes referredto as “segs.” The nucleus of less mature neutrophils is not segmented,but has a band or elongated shape. Less mature neutrophils—those thathave recently been released from the bone marrow into thebloodstream—are known as “bands”. Other data/parameters for a bloodcount can also include, for example, lymphocytes (e.g., increased withsome viral infections such as glandular fever, and in chroniclymphocytic leukemia (CLL), or decreased by HIV infection); monocytes(may be increased in bacterial infection, tuberculosis, malaria, RockyMountain spotted fever, monocytic leukemia, chronic ulcerative colitisand regional enteritis; eosinophil granulocytes (e.g., increased inparasitic infections, asthma, or allergic reaction); basophilgranulocytes (e.g., increased in bone marrow related conditions such asleukemia or lymphoma.

As used herein, data/parameters of a blood count can also include, forexample, data associated with platelets, including platelet numbers,information about their size and the range of sizes in the blood; meanplatelet volume (MPV)—a measurement of the average size of platelets.

In another aspect of the methods of this invention, the cells contactedwith particle contrast agent composition and/or imaged are abnormalcells, such as malaria-infected cells, atypical lymphocytes. In someaspects of this invention, the cells are abnormal cells which can beused to identify, predict, diagnose, prognose, or support a diagnosis ofa condition, disease, infection and/or syndrome.

In another aspect of the methods of this invention, the cells areplatelets.

Unless expressly indicated otherwise, references to “particle” or“particles” made in this disclosure will be understood to encompass anydiscrete or formed object dispersed in a fluid. As used herein,“particle” can include all measurable and detectable (e.g., by imageand/or other measurable parameters) components in biological fluids. Theparticles are of any material, any shape and any size. In certainembodiments, particles can comprise cells. Examples of particles includebut are not limited to cells, including blood cells, fetal cells,epithelials, stem cells, tumor cells, or bacteria, parasites, orfragments of any of the foregoing or other fragments in a biologicalfluid. Blood cells may be any blood cell, including any normal orabnormal, mature or immature cells which potentially exist in abiological fluid, for example, red blood cells (RBCs), white blood cells(WBCs), platelets (PLTs) and other cells. The members also includeimmature or abnormal cells. Immature WBCs may include metamyelocytes,myelocytes, pro-myelocytes and blasts. In addition to mature RBCs,members of RBCs may include nucleated RBCs (NRBCs) and reticulocytes.PLTs may include “giant” PLTs and PLT clumps. Blood cells and formedelements are further described elsewhere in this disclosure.

Exemplary particles can include formed elements in biological fluidsamples, including for example, spherical and non-spherical particles.In certain embodiments, the particles can comprise non-sphericalcomponents. The image projection of non-spherical components can bemaximized in the focal plane of the high optical resolution imagingdevice. In certain embodiments, the non-spherical particles are alignedin the focal plane of the high optical resolution imaging device(aligned in a plane substantially parallel to the direction of theflow). In some embodiments, platelets, reticulocytes, nucleated RBCs,and WBCs, including neutrophils, lymphocytes, monocytes, eosinophils,basophils, and immature WBCs including blasts, promyelocytes,myelocytes, or metamyelocytes are counted and analyzed as particles.

As used herein, detectable and measurable particle parameters caninclude, for example, visual and/or non-image based indices of size,shape, symmetry, contour and/or other characteristics.

In another embodiment, this disclosure relates to a method for imagingparticles using, for example, the kits of this invention, in methodscomprising, for example: 1) illuminating the particles with light in avisual analyzer; 2) obtaining a digitized image of sample particlesenveloped in a PIOAL; and 3) analyzing particle containing samples basedon the image information. In other embodiments, the method may furthercomprise contacting the sample containing particles with a particlecontrast agent composition prior to illuminating the treated sample.

In one embodiment, the particles analyzed comprise at least one of aspherical particle, a non-spherical particle, or both. In anotherembodiment, the particles comprise at least one spherical particle. Instill another embodiment, the particles comprise at least onenonspherical particle. In another embodiment, an image projection ofnon-spherical particles or particles having non-spherical components ismaximized in a plane substantially parallel to the flow direction. Theparticles may be, for example, WBCs, RBCs, and/or platelets. In oneembodiment, at least 50% of the non-spherical particles are aligned in aplane substantially parallel to the direction of flow. In anotheraspect, use of the PIOALs of this invention in a flowcell permits atleast 90% of the non-spherical particles to be aligned in a planesubstantially parallel to the direction of flow.

Flow of the cells smaller than the thickness of the ribbon-shaped samplestream enveloped in PIOAL, results in alignment of those cells parallelto the direction of the flow. In one embodiment of this disclosure, atleast 92% of the non-spherical particles are aligned in a planesubstantially parallel to the direction of flow. In yet anotherembodiment, at least 90% of the non-spherical particles are aligned aplane substantially parallel to the direction of flow. In anotherembodiment, at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94% or at least 95% of the particles are substantially aligned,namely within 20 degrees from a plane substantially parallel to thedirection of flow. In another embodiment, the percentage ofnon-spherical and/or spherical particles are aligned in a planesubstantially parallel to the direction of flow may be any range betweenany two of the recited percentages, for example, at least 75-85%,75-80%, and other ranges such as 75-92%.

Shear forces in the direction parallel to the direction of the flow as aresult of flow of larger cells in the sample enveloped in the PIOAL,such as WBCs, results in positioning, repositioning, and/or betterpositioning of nuclear structures, cytosolic structures or granules orother intracellular components or structures closer to a plane parallelto the direction of the flow

In one embodiment, the non-spherical particles comprise red blood cells.In another aspect of this invention, the spherical particles comprisewhite blood cells or nucleated red blood cells.

In one embodiment of the methods of this invention, the particles arenon-spherical particles. In one embodiment, the particles analyzedcomprise at least one of a spherical particle, a non-spherical particle,or both. In another embodiment, the particles comprise at least onespherical particle. In still another embodiment, the particles compriseat least one nonspherical particle. In another embodiment, an imageprojection of non-spherical particles or particles having non-sphericalcomponents is maximized in a plane substantially parallel to thedirection of flow. The particles may be, for example, RBCs, includingreticulocytes and nucleated RBCs, platelets and/or WBC, including aneutrophil, lymphocyte, monocyte, eosinophil, basophil, or immature WBCincluding a blast, promyelocyte, myelocyte, or metamyelocyte. In oneembodiment, at least 50% of the non-spherical particles are aligned in aplane substantially parallel to the direction of flow. In anotheraspect, use of the PIOALs of this invention in a flowcell permits atleast 90% of the non-spherical particles to be aligned in a planesubstantially parallel to the direction of flow.

In one embodiment of this disclosure, the image cross-section comprisesat least one of differentially stained nuclear structure, differentiallystained cytosolic structure or differentially stained granules in a WBC,including a neutrophil, lymphocyte, monocyte, eosinophil, basophil, orimmature WBC including a blast, promyelocyte, myelocyte, ormetamyelocyte. In another embodiment, at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or at least95% of the spherical and/or non-spherical particles have nuclearstructures, cytosolic structures or granules in the focal plane or depthof field of the high optical resolution imaging device.

In some embodiments of the methods of this invention, the imageinformation is the image cross-section of a particle. In some aspects,the image cross-section comprises at least one of a differentiallystained nuclear structure, a differentially stained cytosolic structureor differentially stained granules in a WBC, including a neutrophil,lymphocyte, monocyte, eosinophil, basophil, or immature WBC including ablast, promyelocyte, myelocyte, or metamyelocyte.

In one embodiment, the methods of this invention provide surprisinglyhigh quality images of cells with a high percentage of particles andparticle content in-focus in flow, which are useful in obtainingautomated, image based WBC differentials, as well as automatedidentification of morphological abnormalities useful in determining,diagnosing, prognosing, predicting, or supporting a diagnosis of whethera subject is healthy or has a disease, condition, abnormality orinfection and/or is responsive or non-responsive to treatment.

In another aspect, the compositions and methods of this inventionprovide more accurate image based cell categorization andsubcategorization and flagging which greatly reduces the manual reviewrate compared to current analyzers.

As used herein, exemplary white blood cells (WBC) can include, forexample, neutrophils, lymphocytes, monocytes, eosinophils, basophils,immature granulocytes including meta-myelocyes, myelocytes,pro-myelocytes and blasts, and abnormal white blood cells. As usedherein, red blood cells (RBC) can include, for example, normal orabnormal red blood cells, reticulocytes, and nucleated red blood cells.

As used herein, viscosity agent can include viscosity agents orviscosity modifiers. An exemplary viscosity agent/modifier has acharacteristic viscosity that is different from the viscosity of thesample such that when the PIOAL and the viscosity agent are mixed, theviscosity of the PIOAL is altered or and/or increased in order tomaximize the alignment of particles. In certain embodiments, theviscosity difference and/or a speed difference between the ribbon-shapedsample stream and the PIOAL can introduce shear forces to act on theparticles while in flow thereby reducing the misalignment and/or causingthe particles to align.

As used herein, the particle contrast agent compositions can be adaptedfor use in combination with a particle and/or intracellular organellealignment liquid (PIOAL) in a visual analyzer for analyzing particles ina sample from a subject. The exemplary PIOAL is useful, as an example,in methods for automated recognition of different types of particles ina sample from a subject.

In another aspect, the cells may be enveloped in PIOAL when images areobtained. Suitable exemplary intracellular organelle alignment liquidsare described herein.

In one embodiment, this disclosure relates to a PIOAL for use in avisual analyzer. In certain embodiments, the PIOAL may comprise at leastone of a buffer; a pH adjusting agent; a buffer; a viscosityagent/modifier; ionic strength modifier, a surfactant, a chelatingagent, and/or an antimicrobial agent.

In one aspect, the PIOAL may comprise two or more viscosityagents/modifiers.

In one aspect, the PIOAL of this invention may have a viscosity ofbetween about 1 to about 10 centipoise. In one embodiment, the PIOAL ofthis invention may comprise a viscosity agent/modifier. In oneembodiment, the PIOAL comprises up to 100% of a viscosity agent.

As used herein, the viscosity agent and/or viscosity modifier caninclude any substance suitable to achieve a viscosity of about 1 toabout 10 centipoise, with optical characteristics, including opticalclarity, appropriate for use in an imaging system. Generally, theviscosity agent or modifier is non-toxic, biocompatible and leaves thecellular structure and contents substantially intact. The viscosityagent and/or viscosity modifier may comprise at least one of glycerol;glycerol derivative; ethylene glycol; propylene glycol(dihydroxypropane); polyethylene glycol; water soluble polymer and/ordextran. In one aspect, the viscosity agent/modifier in the PIOAL may beglycerol. As an example, in one aspect, the viscosity agent/modifier inthe PIOAL may be a glycerol derivative. As an example, in one aspect,the viscosity agent/modifier in the PIOAL may be polyvinylpyrrolidone(PVP). As another example, the viscosity agent/modifier in the PIOAL maybe ethylene glycol. As another example, the viscosity agent/modifier inthe PIOAL may be propylene glycol (dihydroxypropane). As anotherexample, the viscosity agent/modifier in the PIOAL may be polyethyleneglycol. As another example, the viscosity agent/modifier in the PIOALmay be water soluble polymer or dextran. In other aspects, the viscosityagent/modifier in the PIOAL may comprise two or more of glycerol,glycerol derivative; ethylene glycol; propylene glycol(dihydroxypropane); polyvinylpyrrolidone (PVP); polyethylene glycol;water soluble polymer or dextran. Viscosity agent/modifying agents mayinclude any agent suitable to provide a viscosity of about 1 to about 10centipoise, with optical characteristics, including optical clarity,appropriate for use in an imaging system.

As used herein, other exemplary viscosity agents/modifiers can include,for example, natural hydrocolloids (and derivatives), such as Acacia,tragacanth, alginic acid, carrageenan, locust bean gum, guar gum,xanthan gum, gum arabic, guar gum, gelatin, cellulose, alginates,starches, sugars, dextrans; gelatin; sugars (and derivatives), such asdextrose, fructose; polydextrose; dextrans; polydextrans; saccharides;and polysaccharides; semisynthetic hydrocolloids (and derivatives), suchas glycerol, methylcellulose, hydroxyethyl starch (hetastarch), sodiumcarboxymethylcellulose, hydroxyethylcellulose,hydroxypropylmethylcellulose, polyvinylpyrrolidone (PVP); synthetichydrocolloids (and derivatives), such as Polyvinyl alcohol (PVA) and/orCarbopol®. Other cell compatible viscosity agents/modifiers are alsoconsidered useful for this purpose.

In another aspect, the viscosity agent/modifier in the PIOAL may beglycerol present at a concentration of about 1 to about 50% (v/v) of thePIOAL. As an example, in one embodiment, the viscosity agent/modifiermay be present in the PIOAL at a concentration of about 5.0% to about8.0% (v/v). In another aspect, the viscosity agent/modifier may bepresent at a concentration of about 6.5% (v/v). In one embodiment, theviscosity agent/modifier is glycerol present at a concentration of about6.5% (v/v).

In yet another embodiment, the PIOAL can comprise a glycerol viscosityagent/modifier present at a concentration of about 30% (v/v).

In another aspect, the viscosity agent/modifier in the PIOAL may be PVPpresent at a concentration of about 0.5 to about 2.5% (w/v). As anexample, in one embodiment, the viscosity agent/modifier PVP may bepresent in the PIOAL at a concentration of about 1.0 to about 1.6%(w/v). In one embodiment, the PVP is present at a concentration of about1.0% (w/v).

In another aspect, the viscosity agent/modifier in the PIOAL may be PVPand glycerol. As an example, in one embodiment, the glycerol may bepresent in the PIOAL at a concentration of about 5% (v/v) in combinationwith about 1% (w/v) of PVP.

In one embodiment, the PIOAL of this invention may be used in a visualanalyzer to image particles. In one aspect, the visual analyzercomprises a flowcell with a symmetrical flow path, and an autofocuscomponent.

A viscosity agent and/or viscosity modifying/adjusting agents, such asglycerol, may be included in the PIOAL. The viscosity agent, orviscosity modifying agent when introduced, can appropriately adjust theviscosity of the PIOAL to the desired range. Any suitable viscosityagent may be used which sufficiently increases the viscosity of thePIOAL, which has suitable optical characteristics to permit high qualityimaging of cells in flow. The PIOAL will have a suitable viscosity toalign cells and/or cellular structures into a single plane that issubstantially parallel to the direction of the flow, thereby, in part,increasing the in-focus contents of the particles.

The PIOAL may be used with any analyzer of this disclosure.

As used herein, the term “glycerols” encompasses glycerol and aderivative of glycerol (hereinafter referred to as glycerol derivative).Examples of a glycerol derivative include thioglycerol, polyglycerol,and the like. Usable examples of polyglycerol may include diglycerol,POLYGLYCERIN #310 (Sakamoto Yakuhin Kogyo Co., Ltd.), POLYGLYCERIN #750(Sakamoto Yakuhin Kogyo Co., Ltd.), POLYGLYCERIN #500 (Sakamoto YakuhinKogyo Co., Ltd.), and the like.

In another embodiment, the PIOAL of this disclosure further comprises apH adjusting agent. In one aspect, the final pH of the PIOAL and/or thesample is between about 6.0 to about 8.0. In another aspect, the finalpH of the PIOAL and/or the sample is between about 6.6 to about 7.4. Inone aspect, the final pH of the PIOAL may be the same pH as the preparedsample 12B (referring to FIG. 8).

Exemplary pH adjusting agents can include, for example, acids (exemplarsinclude organic acids and mineral acids), bases (exemplars includeorganic bases and hydroxides of alkaline metals and alkaline earthmetals). Exemplary organic acids can include acetic, lactic, formic,citric, oxalic, and uric acids. Exemplary mineral acids can include, forexample, hydrochloric, nitric, phosphoric, sulphuric, boric,hydrofluoric, hydrobromic and perchloric acids. Exemplary organic basescan include, for example, pyridine, methylamine, imidazole,benzimidazole, histidine, phosphazene, and hydroxides of cations.Exemplary hydroxides of alkali metal and alkaline earth metals caninclude, for example, Potassium hydroxide (KOH), Barium hydroxide(Ba(OH)₂), Caesium hydroxide (CsOH), Sodium hydroxide (NaOH), Strontiumhydroxide (Sr(OH)₂), Calcium hydroxide (Ca(OH)₂), Lithium hydroxide(LiOH), and Rubidium hydroxide (RbOH).

In some embodiments, using a buffer, the pH of PIOAL is preferablymaintained from about 6.0 to about 8.5, more preferably from about 7.0to about 8.0. In some embodiments it is preferable to add a buffer agentto the PIOAL in order to adjust the pH of PIOAL. Any suitable bufferagent or agents may be used as long as the agent or agents adjust the pHof the PIOAL to the proper range. Examples of such a buffer agentinclude PBS, Good's buffers (specifically, tris buffer, MES, Bis-Tris,ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, TAPSO, POPSO,HEPPSO, EPPS, Tricine, Bicine, TAPS, and the like), disodiumhydrogenphosphate, sodium dihydrogen phosphate, monobasic potassiumphosphate, veronal sodium-HCl, collidine-HCl,tris(hydroxymethyl)aminomethane-maleic acid,tris(hydroxymethyl)aminomethane-HCl, which may be used alone or incombination.

In another embodiment, the PIOAL of this invention comprises an ionicstrength modifier to adjust the ionic strength of the resultingformulation. Exemplary ionic strength modifiers may include Li⁺, Na⁺,K⁺, Mg⁺⁺, Ca⁺⁺, Cl⁻, Br⁻, HCO⁻ ₃, sulphates, pyrosulphates, phosphates,pyrophosphates (e.g., potassium pyrophosphate), citrates, cacodylates orother suitable salts. In one embodiment, the PIOAL may be isotonic.

Surfactants may be added to the PIOAL. The kinds of surfactants are notparticularly limited as long as they are compatible with othercomponents of the PIOAL, and compatible with the ribbon-shaped samplestream and the particles in the sample. Surfactants may include, forexample, cationic, anionic, nonionic, and ampholytic surfactants.Exemplary surfactants may include polyoxyethylenealkyl ether-typesurfactants, polyoxyethylenealkylphenyl ether-type surfactants, (forexample, NISSAN NONION NS-240 (NOF CORPORATION, registered trademark)),polyoxyethylenesorbitan alkyl ester-type surfactants (for example,RHEODOL TW-0120 (Kao Corporation, registered trademark)), polyolcopolymers (for example, PLURONIC F-127, F-123, F-109, F-87, F-86, F-68,T-1107, T-1102 (BASF Corporation, registered trademark)), MEGA-8,sucrose monocaprate, deoxy-BIGCHAP, n-octyl-β-D-thioglucoside,n-nonyl-β-D-thiomaltoside, n-heptyl-β-D-thioglucoside,n-octyl-β-D-thioglucoside, CHAPS, CHAPSO, and the like may be used.Other surfactants may include Triton-X-100 and Tween 20 at sample andribbon-shaped sample stream compatible concentrations.

The concentration of the surfactant in PIOAL is preferably theconcentration level at which particles such as cells in the sample arenot affected and/or remain substantially intact. Specifically, theconcentration is preferably from 5 to 5000 mg/L, more preferably from100 to 3000 mg/L.

When particles contained in the sample are analyzed with the analyzer,amorphous salts such as ammonium phosphate, magnesium phosphate, calciumcarbonate may precipitate in the sample. Chelating agents may be addedto the PIOAL in order to dissolve these amorphous salts. The addition ofchelating agents enables not only dissolving amorphous salts, but alsoinhibiting the oxidation of PIOAL. Usable examples of a chelating agentinclude EDTA salts, CyDTA, DHEG, DPTA-OH, EDDA, EDDP, GEDTA, HDTA, HIDA,Methyl-EDTA, NTA, NTP, NTPO, EDDPO, and the like. The concentration ofthe chelating agent in the PIOAL is preferable within the range of 0.05to 5 g/L.

In another embodiment, the PIOAL may further comprise one or moreantimicrobial agents. In some aspects, the antimicrobial agent may be,for example, substances which have fungicidal activity (fungicidalagents) and/or substances which have bactericidal activity (bactericidalagents). In certain embodiments, suitable antimicrobial agents caninclude, for example, parabens, isothiazolinone, phenolics, acidicpreservatives, halogenated compounds, quarternia, and alcohol. Exemplaryparabens can include Parabens and Paraben salts. Exemplaryisothiazolinones can include methylchloroisothiazolinone,methylisothiazolinone, benzisothiazolinone ProClin 150, ProClin 200,ProClin 300, and ProClin 950. Exemplary phenolic types can includephenoxyethanol, benzyl alcohol, and phenethyl alcohol. Exemplary acidicpreservatives can include dehydroacetic acid, benzoic acid, sorbic acid,salicylic acid, formic acid, propionic acid. Exemplary halogenatedcompounds can include 2-bromo-2-nitropropane-1, 3-diol, chloroacetamide,chlorobutanol, chloroxylenol, chlorphenesin, dichlorobenzyl alcohol,iodopropynyl butylcarbamate, methyldibromo glutaronitrile. Exemplaryquaternia can include benzalkonium chloride, benzethonium chloride,chlorhexidine, hexamidine diisethionate, and polyaminopropyl biguanide.Exemplary alcohols can include ethyl alcohol and isopropyl alcohol.Examples thereof include triazine antimicrobial agents, thiazolebactericidal agents (for example, benzisothiazolone etc.), pyrithione,pyridine bactericidal agents (for example, 1-hydroxypyridine-2-thiosodium etc.), 2-phenoxyethanol, and the like.Specifically, Proxel GXL (Avecia), TOMICIDE S (API Corporation), and thelike may be used. The bactericidal and/or fungicidal agents help improvethe stability of the PIOAL.

In one embodiment, the concentration of the antimicrobial agent may be0.01% to 0.5% (w/v). The concentration may be 0.03 to 0.05% (w/v).

The sample which is subjected to analysis using the analyzer with thePIOAL in the embodiment is not particularly limited. Samples obtainedfrom the living body (biological samples) are normally used.Alternatively, those samples can be diluted, purified, contacted with acontrast agent, or the like for use. Specifically, examples of such asample may include blood, semen, cerebrospinal fluid, and the like.Samples may also include particle suspensions derived from tissuesamples. The PIOAL in the embodiment is suitably used when particles(red blood cell, white blood cell, bacteria, etc.) are analyzed.

The PIOAL of this invention may be used in a visual analyzer that imagesparticles. In one aspect, the visual analyzer comprises a flowcellcapable of maintaining the flow of a ribbon-shaped sample stream withpredetermined dimensional characteristics, such as an advantageousribbon-shaped sample stream thickness. In some embodiments, the flowcellmay have a symmetrical flow path, and be used in combination with anautofocus component.

This disclosure relates to a method for imaging a particlecomprising: 1) contacting the sample with a particle contrast agentcomposition; 2) illuminating the prepared particle; 3) obtaining adigitized image of the particle in a ribbon-shaped sample streamenveloped in a PIOAL; and; 4) analyzing the image information tocategorize or subcategorize the particles. In some embodiments, theparticle may be at least one of, a WBC, RBC, and/or platelet, including,for example, a neutrophil, lymphocyte, monocyte, eosinophil, basophil,reticulocyte, nucleated RBC, blast, promyelocyte, myelocyte, ormetamyelocyte, cell, bacteria, parasites, particulate matter, cellclump, cellular component, and immature granulocyte. In someembodiments, platelets, reticulocytes, nucleated RBCs, and WBCs,including neutrophils, lymphocytes, monocytes, eosinophils, basophils,and immature WBCs including blasts, promyelocytes, myelocytes, ormetamyelocytes are counted and analyzed based on particle imageinformation.

In some embodiments the visual analyzer comprises a flowcell with asymmetrical or an asymmetrical flowpath, and an autofocus component.

In a general aspect, the exemplary PIOAL and methods of use thereof areuseful when employed in combination with an automated analyzer found inresearch and/or medical laboratories. Exemplary automated analyzers areinstrument designed to measure different formed elements and/or othercharacteristics in a number of biological samples, quickly, including,for example, human body fluid samples, with minimal human assistance.Exemplary automated analyzers can include, for example, hematologyanalyzers and/or cell counters, which can perform for example, completeblood count (CBC) determination. The exemplary analyzers can processsamples singly, in batches, or continuously.

In one aspect, the exemplary analyzer/system comprises an automatedparticle counter configured to detect a plurality of particles that meetone or more selection criteria, and to provide a particle count thereof,wherein the selection criteria encompasses members of at least twocategories within said particles. An analyzer, which may comprise aprocessor, which may include components of the counter, is programmed todistinguish the particles of the at least two categories. A distributionof each of the particles is determined using the analyzer. The processoruses the distribution to correct the particle count for the members ofat least one of the at least two categories and/or subcategories. Insome embodiments, the particle counter comprises at least one channelconfigured to provide the particle count of the at least one categoryand/or subcategory of particles based on a predetermined range based onvolume, size, shape, and/or other criterion. For example, the members ofthe at least one category and/or subcategory comprise at least one typeof particle selected from a group consisting of subcategories of whiteblood cells (WBCs), red blood cells (RBCs), giant platelets (PLTs), andnucleated red blood cells (NRBCs). On a particle counter, due to similarsize or other measured characteristic, cells such as giant PLTs andNRBC's may be counted as WBCs. By operating the apparatus as describedherein, particle count or concentration of giant PLTs and NRBC's can bemeasured accurately.

Each of the calculations or operations described herein may be performedusing a computer or other processor having hardware, software, and/orfirmware. The various method steps may be performed by modules, and themodules may comprise any of a wide variety of digital and/or analog dataprocessing hardware and/or software arranged to perform the method stepsdescribed herein. The modules optionally comprising data processinghardware adapted to perform one or more of these steps by havingappropriate machine programming code associated therewith, the modulesfor two or more steps (or portions of two or more steps) beingintegrated into a single processor board or separated into differentprocessor boards in any of a wide variety of integrated and/ordistributed processing architectures. These methods and systems willoften employ a tangible media embodying machine-readable code withinstructions for performing the method steps described above. Suitabletangible media may comprise a memory (including a volatile memory and/ora non-volatile memory), a storage media (such as a magnetic recording ona floppy disk, a hard disk, a tape, or the like; on an optical memorysuch as a CD, a CD-R/W, a CD-ROM, a DVD, or the like; or any otherdigital or analog storage media), or the like.

All patents, patent publications, patent applications, journal articles,books, technical references, and the like discussed in the instantdisclosure are incorporated herein by reference in their entirety forall purposes.

Different arrangements of the components depicted in the drawings ordescribed above, as well as components and steps not shown or describedare possible. Similarly, some features and sub-combinations are usefuland may be employed without reference to other features andsub-combinations. Embodiments of the invention have been described forillustrative and not restrictive purposes, and alternative embodimentswill become apparent to readers of this patent. In certain cases, methodsteps or operations may be performed or executed in differing order, oroperations may be added, deleted or modified. It can be appreciatedthat, in certain aspects of the invention, a single component may bereplaced by multiple components, and multiple components may be replacedby a single component, to provide an element or structure or to performa given function or functions. Except where such substitution would notbe operative to practice certain embodiments of the invention, suchsubstitution is considered within the scope of the invention.Accordingly, the present invention is not limited to the embodimentsdescribed above or depicted in the drawings, and various embodiments andmodifications can be made without departing from the scope of the claimsbelow.

What is claimed is:
 1. A particle and intracellular organelle alignmentliquid (PIOAL) for use in a combined viscosity and geometrichydrofocusing analyzer, the PIOAL directing flow of a blood sample fluidof a given viscosity that is injected into a narrowing flowcelltransition zone of the visual analyzer so as to produce a sample fluidstream enveloped by the PIOAL, the PIOAL comprising: a fluid having ahigher viscosity than the viscosity of the blood sample fluid, a pHadjusting agent, and Procaine HCl, wherein a viscosity hydrofocusingeffect induced by an interaction between the PIOAL fluid and the samplefluid associated with the viscosity difference, in combination with ageometric hydrofocusing effect induced by an interaction between thePIOAL fluid and the sample fluid associated with the narrowing flowcelltransition zone, is effective to provide a target imaging state in atleast some of the plurality of particles at an imaging site of thevisual analyzer while a viscosity agent in the PIOAL retains viabilityof cells in the sample fluid stream leaving structure and content of thecells intact when the cells extend from the sample fluid stream into theflowing sheath fluid, and wherein the viscosity agent of the sheathfluid comprises glycerol at a concentration between about 1 to about 50%(v/v).
 2. The PIOAL of claim 1, wherein the viscosity agent of thesheath fluid comprises polyvinylpyrrolidone (PVP).
 3. The PIOAL of claim2, wherein the polyvinylpyrrolidone (PVP) is at a concentration of 1%(w/v).
 4. The PIOAL of claim 1, wherein the viscosity agent of thesheath fluid comprises glycerol at a concentration of 5% (v/v) andpolyvinylpyrrolidone (PVP) at a concentration of 1% (w/v).
 5. The PIOALof claim 1, wherein the PIOAL has a viscosity of between about 1-10centipoise (cP).
 6. The PIOAL of claim 1, wherein the viscosity agent ofthe sheath fluid comprises glycerol at a concentration of about 30%(v/v).
 7. The PIOAL of claim 1, wherein the viscosity agent of thesheath fluid comprises glycerol at a concentration of about 6.5% (v/v).8. The PIOAL of claim 1, wherein the pH adjusting agent is sodiumhydroxide, and the PIOAL further comprises sodium sulfate, sodiumchloride, potassium phosphate monobasic, sodium phosphate dibasic,disodium EDTA, and deioinzed water.
 9. The PIOAL of claim 1, wherein thepH of the PIOAL is about 7.2.
 10. The PIOAL of claim 1, wherein thePIOAL is isotonic.
 11. The PIOAL of claim 1, wherein the PIOAL consistsof glycerol, sodium sulfate, sodium chloride, Procaine HCl, potassiumphosphate monobasic, sodium phosphate dibasic, disodium EDTA, deionizedwater, and sodium hydroxide.
 12. The PIOAL of claim 1, furthercomprising a surfactant.
 13. The PIOAL of claim 1, further comprising abuffer.
 14. The PIOAL of claim 1, further comprising an ionic strengthmodifier.
 15. The PIOAL of claim 1, further comprising a chelatingagent.
 16. The PIOAL of claim 1, further comprising an antimicrobialagent.
 17. The PIOAL of claim 1, further comprising a buffer, an ionicstrength modifier, a surfactant, a chelating agent, and an antimicrobialagent.
 18. The PIOAL of claim 1, wherein the PIOAL has a pH equal to thepH of the blood sample fluid.
 19. The PIOAL of claim 1, furthercomprising sodium sulfate, sodium chloride, potassium phosphatemonobasic, sodium phosphate dibasic, disodium EDTA, and deioinzed water.